U.S. patent application number 12/790894 was filed with the patent office on 2011-06-09 for methods and compositions for delivery of catecholic butanes for treatment of tumors.
Invention is credited to Chih-Chuan Chang, Neil Frazer, Jonathan Heller, Ru Chih C. Huang, Yu-Chuan Liang, Elaine Lin, David Mold, Richard Park.
Application Number | 20110135711 12/790894 |
Document ID | / |
Family ID | 36611878 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110135711 |
Kind Code |
A1 |
Huang; Ru Chih C. ; et
al. |
June 9, 2011 |
Methods and Compositions for Delivery of Catecholic Butanes for
Treatment of Tumors
Abstract
The present invention provides kits, methods and compositions
for the treatment of diseases such as cancers. The compositions
herein contain a substantially pure preparation of at least one
catecholic butane, including, for example, NDGA compounds in a
pharmaceutically acceptable carrier or excipient. The catecholic
butane such as NDGA or its derivatives are administered to one or
more subjects in need of treatment by a route other than direct
injection into the affected tissues or topical application on
affected tissues.
Inventors: |
Huang; Ru Chih C.;
(Baltimore, MD) ; Park; Richard; (Ithaca, NY)
; Chang; Chih-Chuan; (Baltimore, MD) ; Liang;
Yu-Chuan; (Baltimore, MD) ; Mold; David;
(Baltimore, MD) ; Lin; Elaine; (New York, NY)
; Heller; Jonathan; (Raleigh, NC) ; Frazer;
Neil; (Cary, NC) |
Family ID: |
36611878 |
Appl. No.: |
12/790894 |
Filed: |
May 31, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11284111 |
Nov 21, 2005 |
7728036 |
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12790894 |
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PCT/US2004/016235 |
May 20, 2004 |
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11284111 |
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60472008 |
May 20, 2003 |
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60472144 |
May 20, 2003 |
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60472188 |
May 20, 2003 |
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60472282 |
May 20, 2003 |
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60472299 |
May 20, 2003 |
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Current U.S.
Class: |
424/450 ;
514/547; 514/551; 514/721; 514/734 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 9/0043 20130101; A61K 31/205 20130101; A61K 9/1075 20130101;
A61K 31/24 20130101; A61P 29/00 20180101; A61K 9/0078 20130101;
A61K 9/0024 20130101; A61K 9/5153 20130101; A61P 31/18 20180101;
A61K 9/0019 20130101; A61K 9/1271 20130101 |
Class at
Publication: |
424/450 ;
514/734; 514/721; 514/547; 514/551 |
International
Class: |
A61K 31/225 20060101
A61K031/225; A61K 31/05 20060101 A61K031/05; A61K 9/127 20060101
A61K009/127; A61K 31/09 20060101 A61K031/09; A61K 31/222 20060101
A61K031/222; A61P 35/00 20060101 A61P035/00; A61P 29/00 20060101
A61P029/00; A61P 31/18 20060101 A61P031/18 |
Claims
1. A method for treatment of a disease in a subject comprising: (a)
providing a composition comprising at least one catecholic butane
other and a pharmaceutically acceptable carrier or excipient,
wherein the wherein the catecholic butane has the formula:
##STR00005## wherein R.sub.1 and R.sub.2 are independently --H, a
lower alkyl, a lower acyl, an alkylene or an unsubstituted or
substituted amino acid residue or salt thereof; R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are
independently --H or a lower alkyl; and R.sub.7, R.sub.8 and
R.sub.9 are independently --H, --OH, a lower alkoxy, a lower
acyloxy, or any two adjacent groups together may be an alkyene
dioxy, or an unsubstituted or substituted amino acid residue or
salt thereof, provided that the catecholic butane is not NDGA. (b)
administering the composition to the subject systemically by a
route of administration selected from the group consisting of: oral
administration; inhalation administration; intra-arterial
administration, with or without occlusion; intracranial
administration; intraventricular administration; intravenous
administration; intramuscular administration; implantation
administration; and central venous administration. (c) wherein the
disease is selected from the group consisting of: acute
lymphoblastic leukemia, acute myeloid leukemia, adrenocortical
carcinoma, anal cancer, astrocytoma, bile duct cancer, bladder
cancer, bone cancer osteosarcoma/malignant fibrous histiocytoma,
brain stem glioma, brain tumor ependymoma, brain tumor
medulloblastoma, breast cancer, carcinoid tumor gastrointestinal,
carcinoma adrenocortical, carcinoma islet cell, cervical cancer,
chronic lymphocytic leukemia, chronic myelogenous leukemia, clear
cell sarcoma of tendon sheaths, colon cancer, cutaneous T-cell
lymphoma, endometrial cancer, epithelial cancer ovarian, esophageal
cancer, Ewing's family of tumors, extragonadal germ cell tumor,
extrahepatic bile duct cancer, eye cancer, intraocular melanoma,
eye cancer retinoblastoma, gallbladder cancer, gastric (stomach)
cancer, gastrointestinal carcinoid tumor, germ cell tumor
extragonadal, germ cell tumor, ovarian tumor, gestational
trophoblastic tumor, glioma, hairy cell leukemia, hepatocellular
(liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer,
intraocular melanoma, islet cell carcinoma (endocrine pancreas),
Kaposi's sarcoma, laryngeal cancer, leukemia acute lymphoblastic
cancer, leukemia acute myeloid cancer, leukemia chronic lymphocytic
cancer, leukemia chronic myelogenous cancer, leukemia hairy cell
cancer, liver cancer, lung cancer non-small cell, lung cancer small
cell, male breast cancer, malignant mesothelioma, medulloblastoma,
melanoma, merkel cell carcinoma, multiple endocrine neoplasia
syndrome, mycosis fungoides, myeloma multiple, nasal cavity,
paranasal and sinus cancer, nasopharyngeal cancer, neuroblastoma,
oral cavity and lip cancer, oropharyngeal cancer,
osteosarcoma/malignant fibrous histiocytoma of bone, ovarian
epithelial cancer, ovarian germ cell tumor, pancreatic cancer,
parathyroid cancer, penile cancer, pheochromocytoma, pineal and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
pleuropulmonary blastoma, prostate cancer, rectal cancer, renal,
pelvis and ureter transitional cell cancer, retinoblastoma,
rhabdomyosarcoma, salivary gland cancer, sarcoma soft tissue adult,
Sezary syndrome, skin cancer, small intestine cancer, stomach
(gastric) cancer, testicular cancer, thymoma, thyroid cancer,
urethral cancer, uterine cancer endometrial, vaginal cancer, vulvar
cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor., other
than an inflammatory disease; other than an inflammatory disease
that is associated with microglial cell activation or stimulation.,
a proliferative disease., a proliferative disease is malignant,
pre-malignant or benign cancer, a disease resulting from or is
associated an infection selected from the group consisting of HIV
infection, HPV infection and HSV infection.
2. The method of claim 1, wherein the composition comprises a
carrier or excipient selected from the group consisting of dimethyl
sulfoxide (DMSO), phosphate buffered saline, saline, a lipid based
formulation, a liposomal formulation, a nanoparticle formulation, a
micellar formulation, a water soluble formulation, and a
biodegradable polymer.
3. The method of claim 1, wherein R.sub.1 and R.sub.2 are
independently --CH.sub.3, --(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or
--(C.dbd.O)CH.sub.2N.sup.+H(CH.sub.3).sub.2.Cl.sup.- and R.sub.8
and R.sub.9 are independently --OCH.sub.3,
--O(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or
--O(C.dbd.O)CH.sub.2N.sup.+H(CH.sub.3).sub.2.Cl.sup.- or wherein
R.sub.1 and R.sub.2 are independently --CH.sub.3 and R.sub.8 and
R.sub.9 are independently --OCH.sub.3.
4. The method of claim 1, wherein the pharmaceutically acceptable
carrier or excipient comprises dimethyl sulfoxide or at least one
dietary fat or oil selected from the group consisting of castor oil
and peanut oil.
5. The method of claim 1, wherein the composition is administered
to a human in an amount of about 10 mg/kg to about 375 mg/kg per
dose.
6. The method of claim 1, wherein the composition is administered
on a schedule selected from the group consisting of: daily, daily
for 5 or more days to a week, daily for 5 or more days to 2 weeks,
daily for 5 or more days to 3 weeks.
7. A method of treating leukemia in a subject comprising: a)
administering a composition consisting essentially of one
catecholic and a pharmaceutically acceptable carrier or excipient,
wherein the wherein the catecholic butane has the formula:
##STR00006## wherein R.sub.1 and R.sub.2 are independently --H, a
lower alkyl, a lower acyl, an alkylene or an unsubstituted or
substituted amino acid residue or salt thereof; R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are
independently --H or a lower alkyl; and R.sub.7, R.sub.8 and
R.sub.9 are independently --H, --OH, a lower alkoxy, a lower
acyloxy, or any two adjacent groups together may be an alkyene
dioxy, or an unsubstituted or substituted amino acid residue or
salt thereof, provided that the catecholic butane is not NDGA; and
(b) administering the composition to the subject systemically by a
route of administration selected from the group consisting of: oral
administration; inhalation administration; intra-arterial
administration, with or without occlusion; intracranial
administration; intraventricular administration; intravenous
administration; intramuscular administration; implantation
administration; and central venous administration.
8. The method of claim 7, wherein R.sub.1 and R.sub.2 are
independently --CH.sub.3, --(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or
--(C.dbd.O)CH.sub.2N.sup.+H(CH.sub.3).sub.2.Cl.sup.- and R.sub.8
and R.sub.9 are independently --OCH.sub.3,
--O(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or
--O(C.dbd.O)CH.sub.2N.sup.+H(CH.sub.3).sub.2.Cl.sup.- or wherein
R.sub.1 and R.sub.2 are independently --CH.sub.3 and R.sub.8 and
R.sub.9 are independently --OCH.sub.3.
9. The method of claim 7 wherein the leukemia is selected from
acute lymphoblastic leukemia, acute myeloid leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia; hairy cell
leukemia; leukemia acute myeloid cancer, leukemia chronic
lymphocytic cancer, leukemia chronic myelogenous cancer, leukemia
hairy cell cancer.
10. The method of claim 7, wherein the pharmaceutically acceptable
carrier or excipient is an oil.
11. The method of claim 7, wherein the composition is administered
on a schedule selected from the group consisting of: daily, daily
for 5 or more days to a week, daily for 5 or more days to 2 weeks,
daily for 5 or more days to 3 weeks.
12. The method of claim 7, wherein the amount of chatecholic butane
administered is at least 30 mg per dose.
13. The method of claim 7 wherein the amount of chatecholic butane
administered is at least 90 mg per dose.
14. The method of claim 7, wherein the chatecholic butane is
present in the composition at a concentration of 20 mg/mL.
15. The method of claim 7, wherein the pharmaceutically acceptable
carrier or excipient comprises Cremaphor EL, ethanol and
saline.
16. The method of claim 8, wherein the Cremaphor EL concentration
is 6%.
17. The method of claim 8, wherein the ethanol concentration is
6%.
18. The method of claim 8, wherein the composition administered to
the subject comprises at least 2 mg of per dose.
19. The method of claim 8, wherein the composition is administered
to a human in an amount of about 10 mg/kg to about 375 mg/kg per
dose.
20. A method of treating leukemia in a subject comprising: a)
administering a composition consisting essentially of
tetra-O-methyl NDGA and a pharmaceutically acceptable carrier or
excipient, and (b) administering the composition to the subject
systemically by a route of administration selected from the group
consisting of: oral administration; inhalation administration;
intra-arterial administration, with or without occlusion;
intracranial administration; intraventricular administration;
intravenous administration; intramuscular administration;
implantation administration; and central venous administration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/284,111 filed Dec. 4, 2007, now U.S. Pat.
No. 7,728,036, which is a continuation of International Patent
Application No. PCT/US2004/016235, filed May 20, 2004, and
published in the English language as International Publication No.
WO 2005/007080 on Jan. 27, 2005, which claims priority to U.S.
provisional application No. 60/472,008, filed May 20, 2003; U.S.
provisional application No. 60/472,144, filed May 20, 2003; U.S.
provisional application No. 60/472,188, filed May 20, 2003; U.S.
provisional application No. 60/472,282, filed May 20, 2003; and
U.S. provisional application No. 60/472,299, filed May 20, 2003;
the contents of all of which are incorporated herein by reference
in their entireties.
BACKGROUND OF THE INVENTION
[0002] This invention relates to kits, methods and compositions
containing catecholic butanes for the delivery of such to subjects
for the treatment of malignant, premalignant and benign tumors.
This invention also relates to methods of making the foregoing
compositions. In such methods of treatment, one or more catecholic
butanes are administered to subjects via routes of delivery other
than direct injection into the affected tissue, and other than
topical application onto the affected tissue. This invention
further relates to compositions comprising one or more catecholic
butanes that are formulated appropriately for such modes of
delivery and treatment.
[0003] Catecholic butanes, including nordihydroguaiaretic acid
("NDGA") and its derivatives, have been used for the inhibition of
tumor growth in certain experimental animals. For example, Jordan
et al. in U.S. Pat. No. 5,008,294 described the use of a single
dose of NDGA on a mammary carcinoma MX-1 xenograft in athymic nude
NCr mice. In one experiment, NDGA was injected into the tumor one
day following subcutaneous implantation of a 14 mg fragment of the
human mammary carcinoma in the axillary region of the mice. Jordan
et al. further described topical application of NDGA after day 23
of implantation of human breast adenocarcinomas in athymic mice.
Some evidence of inhibition of tumor growth was observed in those
experiments, but it is unclear whether the antitumor effect was
durable.
[0004] Huang et al. in U.S. Pat. No. 6,417,234 and U.S. Pat. No.
6,214,874 described intratumor injection of a NDGA derivative,
designated tetra-O-methyl NDGA or M.sub.4N, and another NDGA
derivative, designated G.sub.4N, separately or together into mice
implanted with HPV-16 transformed immortal mouse epithelial cells
(C3). Huang et al. also found some evidence of suppression of tumor
growth by these NDGA derivatives. It is unknown whether compounds
such as these NDGA derivatives can be safely administered to other
animals such as humans.
[0005] Certain of the catecholic butanes, such as M.sub.4N, which
is a NDGA derivative, are hydrophobic compounds found to be soluble
in dimethyl sulfoxide ("DMSO"). When the composition of M.sub.4N in
DMSO was injected into the tumor, the composition appeared to
penetrate most but not all of the tumor tissues. A possible
explanation may be that the hydrophobic nature of the compound
limits its penetration. It would be desirable if a formulation can
be found for safe systemic administration of these hydrophobic
compounds so as to improve their efficacy, expand their utility and
yet maintain their biological activities, such as anti-tumor
activities. It would further be desirable if the catecholic
butanes, including the NDGA Compounds, can be safely administered
by routes of administration other than by direct injection into the
affected tissues or by topical application.
[0006] Moreover, intratumor injection or direct injection of drugs
into affected tissues may not be an ideal treatment regimen.
Patients sometimes experience injection site discomfort. In
addition, many tumors are not amenable to intratumor injection of a
therapeutic, and many may not respond to topical application of a
therapeutic. It would be desirable if a different route of
administration of these catecholic butanes can be found that would
be safe and appropriate for the disease or condition and yet
maintain the biological activities of such compounds.
[0007] Additionally, it is not known whether the catecholic
butanes, including NDGA and NDGA derivatives (collectively, the
"NDGA Compounds"), or formulations containing them can
differentially inhibit the growth or progression of tumor growth in
humans without adversely affecting normal tissues. It would be
desirable if the catecholic butanes, including the NDGA Compounds
can be formulated and administered in such a way as to spare the
normal tissues of any adverse effects.
[0008] Further, a majority of human malignant tumors are both local
and systemic in nature in that the primary malignant tumors are
produced locally whereas the secondary tumors, seeded from the
primary, are spread systemically to other tissues, or arise de novo
from tissues similar to the source of the primary. The most
appropriate therapeutic options, therefore, include those that
deliver effective medication to the primary source of malignancy as
well as to the secondary sources. It would be desirable if an
effective therapeutic can be formulated that can access both the
primary and secondary sources of malignancies.
[0009] It would also be desirable if the NDGA derivatives can be
formulated in a manner that would facilitate delivery to targeted
tissues and maintenance of a certain range of dose level in the
targeted tissues.
BRIEF SUMMARY OF THE INVENTION
[0010] It is, thus, one of the objects of the present invention to
provide methods and compositions for the prevention or treatment of
tumors such as to address the problems in the prior art methods and
compositions, for example, those described in the Background.
[0011] It is another one of the objects of the present invention to
provide methods and compositions as above, such as, for example, to
inhibit the growth, development or progression of tumors.
[0012] It is another one of the objects of the present invention to
provide one or more methods of administering the catecholic
butanes, including the NDGA Compounds, that are effective in the
prevention or treatment of tumors as above, where the targeted
tissues or the affected tissues to be treated are not easily
accessible to direct injection or amenable to topical application
of such compounds.
[0013] It is a further one of the objects of the present invention
to provide one or more formulations containing the catecholic
butanes, including the NDGA Compounds, that can facilitate and/or
optimize distribution of the catecholic butanes, including the NDGA
Compounds, to the targeted tissues.
[0014] It is another one of the objects of the present invention to
provide compositions containing one or more catecholic butanes,
including the NDGA Compounds, in formulations appropriate for
treatment of the targeted tissues.
[0015] In accordance to one of the objects of the present
invention, there is provided a pharmaceutical composition for
treatment of a disease in a subject, such as an animal, for
example, a human, where the composition contains at least one
catecholic butane and a pharmaceutically acceptable carrier or
excipient, and where the composition is formulated for
administration by a route other than by direct injection into or
topical application onto an affected tissue.
[0016] In accordance to another one of the objects, there is
provided a composition as above, where the disease, disorder or
condition is other than an inflammatory disease, for example, other
than an inflammatory disease that is associated with microglial
cell activation or stimulation.
[0017] In accordance to another one of the objects, there is
provided a composition as above, where the disease is a
proliferative disease. Such a proliferative disease may be a
malignant tumor, a premalignant condition, or a benign tumor.
[0018] In accordance to a further one of the objects, there is
provided a composition as above, where the disease results from or
is associated with a virus infection, such as, for example, HIV
infection, HPV infection, or HSV infection.
[0019] In accordance to still another one of the objects, there is
provided a composition as above, where the composition is
formulated for intranasal administration, oral administration,
including through slow release or rapid release capsules, for
inhalation, for subcutaneous administration, for transdermal
administration, for intra-arterial administration, with or without
occlusion, for intracranial administration, intraventricular
administration, intravenous administration, buccal administration,
intraperitoneal administration, intraocular administration, central
venous administration, intramuscular administration or for
implantation.
[0020] In accordance to another one of the objects, there is
provided a composition as above, where the pharmaceutically
acceptable carrier or excipient contains dimethyl sulfoxide (DMSO),
phosphate buffered saline (PBS), saline, an oil such as, for
example, castor oil or corn oil, Cremaphor EL, and ethanol or a
mixture containing one or more of such.
[0021] In accordance to another one of the objects, there is
provided a composition as above, where the pharmaceutically
acceptable carrier or excipient contains a lipid based formulation,
a liposomal formulation, a nanoparticle formulation, a micellar
formulation, a water soluble formulation, a Cremaphor
EL/ethanol/saline formulation or any of the foregoing in a
biodegradable polymer.
[0022] In accordance to yet another one of the objects, there is
provided a composition as above, where the catecholic butane has
the structural formula I as follows:
##STR00001##
where R.sub.1 and R.sub.2 are independently --H, a lower alkyl, a
lower acyl, an alkylene or an unsubstituted or substituted amino
acid residue or salt thereof; R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are independently --H or
a lower alkyl; and R.sub.7, R.sub.8 and R.sub.9 are independently
--H, --OH, a lower alkoxy, a lower acyloxy, or any two adjacent
groups together may be an alkyene dioxy, or an unsubstituted or
substituted amino acid residue or salt thereof.
[0023] In accordance to still another one of the objects, there is
provided a catecholic butane as above, where R.sub.1 and R.sub.2
are independently --H, a lower alkyl, a lower acyl, or an
unsubstituted or substituted amino acid residue or salt thereof;
R.sub.3, R.sub.4, are independently a lower alkyl; R.sub.5,
R.sub.6, R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are
independently --H; and R.sub.7, R.sub.8 and R.sub.9 are
independently --H, --OH, a lower alkoxy, a lower acyloxy, or
unsubstituted or substituted amino acid residue or salt
thereof.
[0024] In accordance to yet another one of the objects, there is
provided a catecholic butane as above, where R.sub.1 and R.sub.2
are independently --H, a lower alkyl, a lower acyl, or an
unsubstituted or substituted amino acid residue or salt thereof;
R.sub.3, R.sub.4, are independently a lower alkyl; R.sub.5,
R.sub.6, R.sub.7, R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are
independently --H; and R.sub.8 and R.sub.9 are independently --OH,
a lower alkoxy, lower acyloxy, or an unsubstituted or substituted
amino acid residue or salt thereof.
[0025] In accordance to still another one of the objects, there is
provided the catecholic butane as above, where R.sub.1 and R.sub.2
are independently --CH.sub.3 or
--(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or a salt thereof.
[0026] In accordance to still another one of the objects, there is
provided the catecholic butane as above, where R.sub.8 and R.sub.9
are independently --OCH.sub.3 or
--O(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or a salt thereof.
[0027] In accordance to still another one of the objects, there is
provided the catecholic butane as above, where R.sub.1 and R.sub.2
are independently --CH.sub.3, --(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2
or --(C.dbd.O)CH.sub.2N.sup.+H(CH.sub.3).sub.2.Cl.sup.- and R.sub.8
and R.sub.9 are independently --OCH.sub.3,
--O(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or
--O(C.dbd.O)CH.sub.2N.sup.+H(CH.sub.3).sub.2.Cl.sup.-.
[0028] In accordance to still another one of the objects, there is
provided the catecholic butane as above, where R.sub.1 and R.sub.2
are independently --H or --CH.sub.3 and R.sub.8 and R.sub.9 are
independently --OH or --OCH.sub.3, provided that the catecholic
butane is not NDGA.
[0029] In accordance to still another one of the objects, there is
provided the catecholic butane as above, where R.sub.1 and R.sub.2
are independently --CH.sub.3 and R.sub.8 and R.sub.9 are
independently --OCH.sub.3.
[0030] In accordance to still another one of the objects, there is
provided the catecholic butane as above, where the catecholic
butane is NDGA.
[0031] In accordance to still another one of the objects, there is
provided the catecholic butane as above, where the catecholic
butane is other than NDGA.
[0032] In accordance to yet another one of the objects, there is
provided a method of making a pharmaceutical composition containing
a catecholic butane, where the method includes the steps of (a)
providing a catecholic butane as above; (b) providing a
pharmaceutically acceptable carrier or excipient as above, and (c)
combining the catecholic butane with the pharmaceutically
acceptable carrier or excipient.
[0033] In accordance to a further one of the objects of the present
invention, there is provided a method of treating a disease in a
subject, where the method of treatment includes providing a
pharmaceutical composition as above and administering the
composition to the subject by a route other than by direct
injection into the tumor or topical application onto the tumor.
[0034] In accordance to another one of the objects, there is
provided a method of treatment as above, where the disease is other
than an inflammatory disease, for example, other than an
inflammatory disease that is associated with microglial cell
activation or stimulation.
[0035] In accordance to another one of the objects, there is
provided a method of treatment as above, where the disease is a
proliferative disease such as a malignant tumor, a premalignant
condition, or a benign tumor.
[0036] In accordance to a further one of the objects, there is
provided a method of treatment as above, where the disease results
from or is associated with a virus infection, such as, for example,
HIV infection, HPV infection, or HSV infection.
[0037] In accordance to still another one of the objects, there is
provided a method of treatment as above, where the composition is
formulated for intranasal administration, oral administration,
including through slow release or rapid release capsules, for
inhalation, for subcutaneous administration, for transdermal
administration, for intra-arterial administration, with or without
occlusion, for intracranial administration, intraventricular
administration, intravenous administration, buccal administration,
intraperitoneal administration, intraocular administration, central
venous administration, intramuscular administration or for
implantation.
[0038] In accordance to another one of the objects, there is
provided a method of treatment as above, where the pharmaceutically
acceptable carrier or excipient contains dimethyl sulfoxide (DMSO),
phosphate buffered saline (PBS), saline, an oil such as, for
example, castor oil or corn oil, Cremaphor EL, ethanol and any
combination of such.
[0039] In accordance to another one of the objects, there is
provided a method of treatment as above, where the pharmaceutically
acceptable carrier or excipient contains a lipid based formulation,
a liposomal formulation, a nanoparticle formulation, a micellar
formulation, a water soluble formulation, a Cremaphor
EL/ethanol/saline formulation or any of the foregoing in a
biodegradable polymer.
[0040] In accordance to yet another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane has a formula given above.
[0041] In accordance to yet another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is tetra-O-methyl NDGA.
[0042] In accordance to still another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is tetra-dimethylglycinyl NDGA.
[0043] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is tri-O-methyl NDGA.
[0044] In accordance to still another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is NDGA.
[0045] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is other than NDGA.
[0046] In accordance to another one of the objects, there is
provided a method of treatment as above, where the method includes
administering at least two catecholic butanes.
[0047] In accordance to another one of the objects, there is
provided a method of treatment as above, where the two catecholic
butanes are administered substantially contemporaneously.
[0048] In accordance to another one of the objects, there is
provided a method of treatment as above, where the two catecholic
butanes are administered at different times.
[0049] In accordance to another one of the objects, there is
provided a method of treatment as above, where the two catecholic
butanes are selected from the group consisting of tetra-O-methyl
NDGA, tri-O-methyl NDGA and tetra-dimethylglycinyl NDGA.
[0050] In accordance to another one of the objects, there is
provided a method of treatment as above, where the nanoparticle
formulation contains at least one selected from the group
consisting of poly(DL-lactide-co-glycolide), poly vinyl alcohol,
d-.alpha.-tocopheryl polyethylene glycol 1000 succinate, and
poly(lactide-co-glycolide)-monomethoxy-poly(polyethylene
glycol).
[0051] In accordance to another one of the objects, there is
provided a method of treatment as above, where the liposomal
formulation comprises at least one selected from the group
consisting of phosphatidylcholine/cholesterol/PEG-DPPE,
distearoylphosphatidylcholine/cholesterol/PEG-DPPE, and
1-2-dioleoyl-sn-glycero-3-phosphocholine/1-2-dipalmitoyl-sn-glycero-3-pho-
spho-rac-(1-glycerol) sodium
salt/cholesterol/triolein/tricaprylin.
[0052] In accordance to another one of the objects, there is
provided a method of treatment as above, where the disease is
cancer and the cancer is a solid tumor, a lymphoma or leukemia.
[0053] In accordance to another one of the objects, there is
provided a method of treatment as above, where the cancer is
selected from the group consisting of malignant, pre-malignant or
benign brain tumor, nasal pharyngeal tumor, head and neck tumor,
liver tumor, kidney tumor, prostate tumor, breast tumor, a bladder
tumor, pancreatic tumor, stomach tumor, colon tumor, ovarian tumor,
cervical tumor, and skin tumor and metastases thereto.
[0054] In accordance to another one of the objects, there is
provided a method of treatment as above, where the method comprises
administering the composition more than once.
[0055] In accordance to another one of the objects, there is
provided a method of treatment as above, where the pharmaceutically
acceptable carrier or excipient is an aqueous preparation.
[0056] In accordance to another one of the objects, there is
provided a method of treatment as above, where the pharmaceutically
acceptable carrier or excipient comprises a hydrophobic
preparation.
[0057] In accordance to another one of the objects, there is
provided a method of treatment as above, where the hydrophobic
preparation comprises a lipid based vehicle.
[0058] In accordance to another one of the objects, there is
provided a method of treatment as above, where the pharmaceutically
acceptable carrier or excipient comprises at least one selected
from the group consisting of castor oil, peanut oil, dimethyl
sulfoxide (DMSO), and other dietary fats or oils.
[0059] In accordance to another one of the objects, there is
provided a method of treatment as above, where the composition is
formulated in the form of one selected from the group consisting of
a tablet, a powder, a gel capsule, a liquid, and an oral rinse.
[0060] In accordance to another one of the objects, there is
provided a method of treatment as above, where the pharmaceutically
acceptable carrier or excipient comprises a polymer
formulation.
[0061] In accordance to another one of the objects, there is
provided a method of treatment as above, where the polymer
formulation is a biodegradable polymer formulation.
[0062] In accordance to another one of the objects, there is
provided a method of treatment as above, where the pharmaceutically
acceptable carrier or excipient allows for high local drug
concentration and sustained release over a period of time.
[0063] In accordance to another one of the objects, there is
provided a method of treatment as above, where the polymer
formulation comprises at least one selected from the group
consisting of 1,3-bis(p-carboxyphenoxy) propane, sebacic acid,
poly(ethylene-co-vinyl acetate), and
poly(lactide-co-glycolide).
[0064] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is dissolved in saline, DMSO or ethanol prior to
administration.
[0065] In accordance to another one of the objects, there is
provided a method of treatment as above, where the composition is
at least one selected from the group consisting of: a powder, an
aerosol, an aqueous formulation, a liposomal formulation, a
nanoparticle formulation, and a hydrophobic formulation.
[0066] In accordance to another one of the objects, there is
provided a method of treatment as above, where the composition is
administered daily for a defined period of time.
[0067] In accordance to another one of the objects, there is
provided a method of treatment as above, where the composition is
administered intermittently.
[0068] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is infused into the subject.
[0069] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is a water soluble compound.
[0070] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is a hydrophobic compound.
[0071] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is formulated as a liquid, an aerosol, an oral rinse, a
suspension, a tablet, a powder, or a gel capsule.
[0072] In accordance to yet one of the objects, there is provided a
method of treatment of a viral infection in a subject comprising
administering the composition of claim 1 to the subject, wherein
the viral infection results from or is associated with HIV, HPV, or
HSV.
[0073] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is administered in a range of greater than about 10 mg/kg
and less than about 375 mg/kg per dose into humans.
[0074] In accordance to another one of the objects, there is
provided a method of treatment as above, where the range is greater
than about 10 mg/kg and less than about 250 mg/kg per dose.
[0075] In accordance to another one of the objects, there is
provided a method of treatment as above, where the range is greater
than about 10 mg/kg and less than about 200 mg/kg per dose.
[0076] In accordance to another one of the objects, there is
provided a method of treatment as above, where the range is greater
than about 10 mg/kg and less than about 150 mg/kg per dose.
[0077] In accordance to another one of the objects, there is
provided a method of treatment as above, where the range is greater
than about 10 mg/kg and less than about 100 mg/kg per dose.
[0078] In accordance to another one of the objects, there is
provided a method of treatment as above, where the range is greater
than about 10 mg/kg and less than about 75 mg/kg per dose.
[0079] In accordance to another one of the objects, there is
provided a method of treatment as above, where the range is greater
than about 10 mg/kg and less than about 50 mg/kg per dose.
[0080] In accordance to another one of the objects, there is
provided a method of treatment as above, where the composition is
administered systemically, such as intravenously, for example.
[0081] In accordance to another one of the objects, there is
provided a method of treatment as above, where the catecholic
butane is tri-O-NDGA or tetra-O-methyl NDGA.
[0082] In accordance to still one of the objects, there is provided
a kit for treatment of a disease comprising the pharmaceutical
composition above and instructions for administration of the
composition.
[0083] In accordance to a further one of the objects, there is
provided a method of treating a tumor in a subject, where the tumor
is a malignant, premalignant or benign tumor, and where the tumor
arises from or is associated with a tissue or organ selected from
the group consisting of: breast, liver, stomach, pancreas,
colorectal, colon and prostate, comprising the steps of: (a)
providing a composition containing tetra-O-methyl NDGA (M.sub.4N)
and a pharmaceutically acceptable carrier or excipient; and (b)
administering the composition to the subject; where the composition
is administered other than by direct injection into or topical
application onto the tumor.
[0084] In accordance to a further one of the objects, there is
provided a method of treating a tumor as above, where the method
includes administering the composition orally, where the oral
composition may be a slow release formulation or a rapid release
formulation.
[0085] In accordance to a further one of the objects, there is
provided a method of treating a tumor as above, where the
pharmaceutically acceptable carrier or excipient is an oil, such
as, for example, castor oil or corn oil.
[0086] In accordance to a further one of the objects, there is
provided a method of treating a tumor as above, where the
composition is present in an edible mix.
[0087] In accordance to a further one of the objects, there is
provided a method of treating a tumor as above, where the
catecholic butane composition is administered daily for a period of
time, such as, for example, daily for 5 or more days to a week, or
daily for 5 or more days to 2 weeks, or daily for 5 or more days to
3 weeks.
[0088] In accordance to a further one of the objects, there is
provided a method of treating a tumor as above, where the amount of
tetra-O-methyl NDGA administered is at least 30 mg per dose, or
optionally, at least 90 mg per dose.
[0089] In accordance to a still further one of the objects, there
is provided a method of treating a tumor as above, where
tetra-O-methyl NDGA is present in the composition at a
concentration of 20 mg/mL.
[0090] In accordance to a further one of the objects, there is
provided a method of treating a tumor as above, where the
pharmaceutically acceptable carrier or excipient comprises
Cremaphor EL, ethanol and saline, where Cremaphor EL may be present
at a concentration of about 6%, ethanol may be present at a
concentration of about 6%, and saline may be present at a
concentration of about 88%, for example.
[0091] In accordance to another one of the objects, there is
provided a method of treating a tumor as above, where the
composition administered to the subject comprises at least 2 mg of
tetra-O-methyl NDGA per dose.
[0092] In accordance to a further one of the objects, there is
provided a method of treating a tumor as above, where the
composition is administered intravenously or intraperitoneally.
[0093] In accordance to a still another one of the objects, there
is provided a method of treating a tumor as above, where the
composition is administered more frequently than once every 6 days
for a period of time or optionally, more frequently than once every
2 days for a period of time.
[0094] Further objects, features and advantages of the present
invention will be apparent to one of ordinary skill in the art upon
reading the present description. Such other objects, features, and
advantages are also deemed embodied by the present invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0095] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings.
[0096] In the drawings:
[0097] FIG. 1 shows systemic distribution of M.sub.4N to various
organs at 3 hours following intravenous and intraperitoneal
injection. Mice were injected with 100 .mu.Ci of .sup.3H-- M.sub.4N
and 60 mM of unlabeled M.sub.4N. Organs and blood were harvested
and weighed at 3 hours post-injection and the M.sub.4N was
extracted. The tritium content of the organ extracts were measured,
and the quantity of M.sub.4N in each organ was calculated based on
the specific activity of the inoculum. FIG. 1A represents the
quantity of M.sub.4N, in micrograms per gram of tissue, found in
each organ containing a relatively high quantity of M.sub.4N. FIG.
1B represents those organs containing a relatively low quantity of
M.sub.4N.
[0098] FIG. 2 shows systemic tissue distribution profile of
M.sub.4N at various time points. Mice were injected with 100 .mu.Ci
of .sup.3H-- M.sub.4N and 60 mM of unlabeled M.sub.4N. Organs and
blood were harvested and weighed at 4, 6, 18 hours and 6 days
post-injection and the M.sub.4N was extracted. The tritium content
of the organ extracts were measured, and the quantity of M.sub.4N
in each organ was calculated based on the specific activity of the
inoculum.
[0099] FIG. 3 shows the body weights of mice during long-term oral
feeding of M.sub.4N, indicating no apparent toxicity. Male and
female mice were continually fed food balls weighing 9 g and
containing 280 mg of M.sub.4N for 14 weeks. On average, mice
consumed 93.3 mg of M.sub.4N per day. Control mice were fed food
balls containing no M.sub.4N. Body weights were recorded
periodically.
[0100] FIG. 4 shows that systemic treatment with M.sub.4N inhibits
the in vivo growth of human tumor xenografts. Athymic nude mice
were implanted s.c. in each flank with MCF-7 breast adenocarcinoma
cells, Hep3B hepatocellular carcinoma cells, HT-29 colorectal
carcinoma cells, and LNCaP prostate carcinoma cells. When tumors
attained a mean diameter of 7-8 mm, mice received for three weeks a
single daily i.p. injection containing 2 mg of M.sub.4N dissolved
in 100 .mu.L Cremaphor-ethanol based solvent. Control mice received
vehicle only. Tumors were measured in two perpendicular dimensions
(L and W) once every seven days, and tumor volumes were calculated
according to the formula: V=(L.times.W/2).sup.3.times..pi./6.
[0101] FIG. 5 shows the serum concentration of M.sub.4N in dogs
given different doses of M.sub.4N at different time points.
[0102] FIG. 6 is a schematic representation of examples of
different modes of delivery of the NDGA derivatives to the brain
for treatment of brain tumors. M.sub.4N represents a hydrophilic
NDGA and G.sub.4N represents a lipophilic NDGA. OD represents
osmotic disruption of blood brain barrier. SC represents
subcutaneous administration. IP represents intraperitoneal
administration. IM represents intramuscular administration.
[0103] FIG. 7 is a schematic representation of examples of
different modes of delivery of the NDGA derivatives to tissues
other than the brain for the treatment of tumors. M.sub.4N
represents a hydrophilic NDGA and G.sub.4N represents a lipophilic
NDGA. SC represents subcutaneous administration. IP represents
intraperitoneal administration. IM represents intramuscular
administration.
[0104] Table 1. Oral administration of M.sub.4N results in systemic
tissue distribution. (A) Short-term oral feeding of M.sub.4N. Three
mice were fed 30 mg of M.sub.4N dissolved in castor oil. At 2, 4,
and 8 hours post-feeding, tissues were removed and weighed. The
quantity of M.sub.4N present in tissues was then quantitated by
HPLC. (B) Long-term oral feeding of M.sub.4N. Mice were continually
fed food balls weighing 9 g and containing 280 mg of M.sub.4N for
14 weeks. On average, mice consumed 93.3 mg of M.sub.4N per day.
The quantity of M.sub.4N present in tissues was quantitated by
HPLC.
[0105] Table 2. Systemic treatment with M.sub.4N inhibits the in
vivo growth of human tumor xenografts. Athymic nude mice were
implanted s.c. in each flank with MCF-7 breast adenocarcinoma
cells, Hep3B hepatocellular carcinoma cells, HT-29 colorectal
carcinoma cells, and LNCaP prostate carcinoma cells. When tumors
attained a mean diameter of 7-8 mm, mice received for three weeks a
single daily i.p. injection containing 2 mg of M.sub.4N dissolved
in 100 .mu.L Cremaphor-ethanol based solvent. Control mice received
vehicle only. Tumors were measured in two perpendicular dimensions
(L and W) once every seven days, and tumor volumes were calculated
according to the formula: V=(L.times.W/2).sup.3.times..pi./6.
[0106] Table 3. Tumor size change for all tumors following 21 days
of treatment. Athymic nude mice were implanted s.c. in each flank
with MCF-7 breast adenocarcinoma cells, Hep3B hepatocellular
carcinoma cells, HT-29 colorectal carcinoma cells, and LNCaP
prostate carcinoma cells. When tumors attained a mean diameter of
7-8 mm, mice received for three weeks a single daily i.p. injection
containing 2 mg of M.sub.4N dissolved in 100 .mu.L
Cremaphor-ethanol based solvent. Control mice received vehicle
only. Tumors were measured in two perpendicular dimensions (L and
W) once every seven days, and tumor volumes were calculated
according to the formula: V=(L.times.W/2).sup.3.times..pi./6.
DETAILED DESCRIPTION OF THE INVENTION
[0107] The inventors herein have discovered that catecholic butanes
of the formula I:
##STR00002##
where R.sub.1 and R.sub.2 are independently --H, a lower alkyl, a
lower acyl, an alkylene or an unsubstituted or substituted amino
acid residue or salt thereof; R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are independently --H or
a lower alkyl; and R.sub.7, R.sub.8 and R.sub.9 are independently
--H, --OH, a lower alkoxy, a lower acyloxy, or any two adjacent
groups together may be an alkyene dioxy, or an unsubstituted or
substituted amino acid residue or salt thereof are useful for the
treatment of proliferative diseases such as cancer, when applied
other than by direct injection into the tumor or topically onto the
situs of the tumor. Such catecholic butanes can be combined with
pharmaceutically acceptable carrier or excipient to produce
pharmaceutical compositions that can be formulated for different
routes of delivery.
[0108] In another embodiment of the invention, the catecholic
butane has the formula above where R.sub.1 and R.sub.2 are
independently --H, a lower alkyl, a lower acyl, or an unsubstituted
or substituted amino acid residue or salt thereof; R.sub.3,
R.sub.4, are independently a lower alkyl; R.sub.5, R.sub.6,
R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are independently --H;
and R.sub.7, R.sub.8 and R.sub.9 are independently --H, --OH, a
lower alkoxy, a lower acyloxy, or an unsubstituted or substituted
amino acid residue or salt thereof.
[0109] In a further embodiment of the invention, the pharmaceutical
composition has the above formula where R.sub.1 and R.sub.2 are
independently --H, a lower alkyl, a lower acyl, or an unsubstituted
or substituted amino acid residue or salt thereof; R.sub.3,
R.sub.4, are independently a lower alkyl; R.sub.5, R.sub.6,
R.sub.7, R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are
independently --H; and R.sub.8 and R.sub.9 are independently --OH,
a lower alkoxy, lower acyloxy, or an unsubstituted or substituted
amino acid residue or salt thereof.
[0110] In a further embodiment of the invention, the pharmaceutical
composition has the formula above where R.sub.1 and R.sub.2 are
independently --CH.sub.3 or --(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or
a salt thereof.
[0111] In another embodiment of the invention, the pharmaceutical
composition has the formula above where R.sub.8 and R.sub.9 are
independently --OCH.sub.3 or --O(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2
or a salt thereof.
[0112] In a further embodiment of the invention, the pharmaceutical
composition has the formula above where R.sub.1 and R.sub.2 are
independently --CH.sub.3, --(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or
--(C.dbd.O)CH.sub.2N.sup.+H(CH.sub.3).sub.2.Cl.sup.- and R.sub.8
and R.sub.9 are independently --OCH.sub.3,
--O(C.dbd.O)CH.sub.2N(CH.sub.3).sub.2 or
--O(C.dbd.O)CH.sub.2N.sup.+H(CH.sub.3).sub.2.Cl.sup.-.
[0113] In yet another embodiment of the invention, the
pharmaceutical composition has the formula above where R.sub.1 and
R.sub.2 are independently --H or --CH.sub.3 and R.sub.8 and R.sub.9
are independently --OH or --OCH.sub.3, provided that the catecholic
butane is not NDGA.
[0114] In a different embodiment of the invention, the
pharmaceutical composition has the formula as above where R.sub.1
and R.sub.2 are independently --CH.sub.3 and R.sub.8 and R.sub.9
are independently --OCH.sub.3.
[0115] In yet another embodiment of the invention, the catecholic
butane is NDGA. In an alternative embodiment, the catecholic butane
is other than NDGA, namely, a NDGA derivative with the following
formula II:
##STR00003##
[0116] The present inventors have surprisingly discovered that a
composition containing a substantially pure preparation of at least
one NDGA derivative is effective for the treatment of proliferative
diseases such as tumors, when such composition is administered via
a route other than the direct injection into the affected or target
tissues, and other than by topical application onto the affected
tissue. The NDGA derivatives herein have a formula II as set forth
above, where R.sub.1, R.sub.2, R.sub.3 and R.sub.4 independently
represent --OH, a lower alkoxy, for example, --OCH.sub.3, a lower
acyloxy, for example, --O(C.dbd.O)CH.sub.3, or unsubstituted or
substituted amino acid residue or salt thereof but are not each
--OH simultaneously; and R.sub.5, R.sub.6 independently represent
--H or an alkyl such as a lower alkyl, for example, --CH.sub.3 or
--CH.sub.2CH.sub.3. In one embodiment, R.sub.5 and R.sub.6 can both
be --H, --CH.sub.3 or --CH.sub.2CH.sub.3.
[0117] The present catecholic butane, including the NDGA Compounds,
in a suitable formulation, can be safely administered to one or
more subjects in need of such treatment by intranasal delivery.
Optionally, such catecholic butanes or NDGA Compounds can be
administered by inhalation. Further optionally, such catecholic
butanes or NDGA Compounds can be administered orally, such as by
mixing with food, for example, or buccally, or intraocularly.
Additionally, the catecholic butanes or NDGA Compounds can be
administered as an oral rinse, for example, in a rinse-and-spit
treatment one or more times a day.
[0118] Moreover, the catecholic butanes or NDGA Compounds
formulated in liposomal formulations, nanoparticle formulations, or
micellar formulations can additionally be safely administered
systemically, such as intravenously, such as by injection into the
central vein for example, or intraperitoneally, interstitially,
subcutaneously, transdermally, intramuscularly, intra-arterially,
intra-cranially, or intra-ventricularly.
[0119] Furthermore, the catecholic butanes or NDGA Compounds can be
formulated in liposomal formulations, nanoparticles formulations,
or micellar formulations, or any formulation embedded in a
biodegradable polymer, for administration into a subject, such as
one in need of such treatment. Implantation into the brain, for
example, can be used for treatment of brain tumors.
[0120] In one embodiment of the invention, the route of
administration for purposes herein is other than by parenteral
administration, where parenteral administration herein means
intravenous, intra-arterial, intramuscular, subcutaneous,
transdermal and intraperitoneal administration.
[0121] The present invention further features a pharmaceutical
composition containing catecholic butanes or NDGA Compounds for
treatment of proliferative diseases such as tumors where the
composition is formulated for delivery or administration as
described above such as, for example, in the form of a tablet, a
liquid that is either hydrophilic or hydrophobic, a powder such as
one resulting from lyophilization, an aerosol, or in the form of an
aqueous water soluble composition, a hydrophobic composition, a
liposomal composition, a micellar composition, such as that based
on Tween 80 or diblock copolymers, a nanoparticle composition, a
polymer composition, a cyclodextrin complex composition, emulsions,
lipid based nanoparticles termed "lipocores."
[0122] The present invention further features a method of producing
the pharmaceutical composition of the present invention, the method
involving making or providing the catecholic butanes or NDGA
Compounds in a substantially purified form, combining the
composition with a pharmaceutically acceptable carrier or
excipient, and formulating the composition in a manner that is
compatible with the mode of desired administration.
[0123] In a further aspect of the present invention, there is
provided a method of treating tumor as above, where the tumor is
selected from the group consisting of lung, prostate, breast,
colon, liver, kidney, ovarian, cervical, skin, pancreas, brain,
leukemias, lymphomas, gastrointestinal tumor such as stomach, soft
tissue sarcomas and the like.
[0124] The present invention still additionally provides for kits
comprising compositions or formulations as above for the treatment
of proliferative diseases such as tumors where the compositions are
formulated for delivery as above, including but not limited to
intranasal administration, inhalation, oral administration,
intravenous administration, intraperitoneal administration and
other parenteral administration, or as an oral rinse, or the like,
and instructions for such administration.
DEFINITIONS
[0125] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
present invention may be better understood in light of the
particular meanings as follows.
[0126] The term "active agent," "compound," and "drug" herein
refers to one or more catecholic butanes, including NDGA and NDGA
derivatives.
[0127] The term "alkylene dioxy" as used herein refers to methylene
(or substituted methylene) dioxy or ethylene (or substituted
ethylene) dioxy.
[0128] The term "unsubstituted or substituted amino acid residue or
salt thereof" in reference to one of the R groups in the formula
for the catecholic butane herein is an amino acid residue or a
substituted amino acid residue or salt of an amino acid residue or
salt of a substituted amino acid residue including but not limited
to: alanine, arginine, asparagine, aspartate, cysteine, glutamate,
glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, valine, 5-hydroxylysine, 4-hydroxyproline, thyroxine,
3-methylhistidine, .epsilon.-N-methyllysine,
.epsilon.-N,N,N-trimethyllysine, aminoadipic acid,
.gamma.-caroxyglutamic acid, phosphoserine, phosphothreonine,
phosphotyrosine, N-methylarginine, N-acetyllysine, and an
N,N-dimethyl-substituted amino acid residue; or a salt thereof,
such as a chloride salt.
[0129] The term "lower alkyl" means C.sub.1-C.sub.6 alkyl.
[0130] The term "lower acyl" means C.sub.1-C.sub.6 acyl.
[0131] The term "NDGA Compound" refers to NDGA and/or its
derivatives, singly or collectively.
[0132] The term "NDGA derivative" refers to a derivative of NDGA
each having the formula II:
##STR00004##
wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently
--OH, lower alkoxy, lower acyloxy, or an unsubstituted or
substituted amino acid residue or salt thereof but are not each
--OH simultaneously; and R.sub.5, R.sub.6 are independently --H or
an alkyl such as a lower alkyl. The term includes, for example, a
compound in which R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each
--OCH.sub.3, or are each --O(C.dbd.O)CH.sub.3; and R.sub.5, R.sub.6
are each --H or each a lower alkyl. In one embodiment of the
invention, R.sub.5, R.sub.6 are each --CH.sub.3 or
--CH.sub.2CH.sub.3.
[0133] A "substantially purified" compound in reference to the
catecholic butanes or NDGA Compounds herein is one that is
substantially free of compounds that are not the catecholic butane
or NDGA Compounds of the present invention (hereafter, "non-NDGA
materials"). By substantially free is meant at least 50%,
preferably at least 70%, more preferably at least 80%, and even
more preferably at least 90% free of non-NDGA materials.
[0134] The "buffer" suitable for use herein includes any buffer
conventional in the art, such as, for example, Tris, phosphate,
imidazole, and bicarbonate.
[0135] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a condition or disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete cure
for a condition or disease and/or adverse affect attributable to
the condition or disease. "Treatment," thus, for example, covers
any treatment of a condition or disease in a mammal, particularly
in a human, and includes: (a) preventing the condition or disease
from occurring in a subject which may be predisposed to the
condition or disease but has not yet been diagnosed as having it;
(b) inhibiting the condition or disease, such as, arresting its
development; and (c) relieving, alleviating or ameliorating the
condition or disease, such as, for example, causing regression of
the condition or disease.
[0136] A "pharmaceutically acceptable carrier" refers to a
non-toxic solid, semisolid or liquid filler, diluent, encapsulating
material or formulation auxiliary of any conventional type. A
"pharmaceutically acceptable carrier" is non-toxic to recipients at
the dosages and concentrations employed, and is compatible with
other ingredients of the formulation. For example, the carrier for
a formulation containing the present catecholic butane or NDGA
Compounds preferably does not include oxidizing agents and other
compounds that are known to be deleterious to such. Suitable
carriers include, but are not limited to, water, dextrose,
glycerol, saline, ethanol, buffer, dimethyl sulfoxide, Cremaphor
EL, and combinations thereof. The carrier may contain additional
agents such as wetting or emulsifying agents, or pH buffering
agents. Other materials such as anti-oxidants, humectants,
viscosity stabilizers, and similar agents may be added as
necessary.
[0137] Pharmaceutically acceptable salts herein include the acid
addition salts (formed with the free amino groups of the
polypeptide) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, mandelic, oxalic, and tartaric. Salts formed with the free
carboxyl groups may also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, and histidine.
[0138] The term "pharmaceutically acceptable excipient," includes
vehicles, adjuvants, or diluents or other auxiliary substances,
such as those conventional in the art, which are readily available
to the public. For example, pharmaceutically acceptable auxiliary
substances include pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like.
[0139] The terms "subject," "host," and "patient," are used
interchangeably herein to refer to an animal being treated with the
present compositions, including, but not limited to, simians,
humans, felines, canines, equines, bovines, porcines, ovines,
caprines, mammalian farm animals, mammalian sport animals, and
mammalian pets.
[0140] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0141] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0142] All publications mentioned herein, including patents, patent
applications, and journal articles are incorporated herein by
reference in their entireties including the references cited
therein, which are also incorporated herein by reference.
[0143] It must be noted that as used herein, the singular forms
"a", "an", and "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, reference to "a
compound" includes a plurality of such compounds and reference to
"the NDGA Compound" includes reference to one or more NDGA
Compounds and equivalents thereof known to those skilled in the
art.
[0144] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
[0145] The invention described below is given by way of example
only and is not to be interpreted in any way as limiting the
invention.
[0146] Preparation of Catecholic Butanes
[0147] The catecholic butanes of the present invention can be
prepared by any conventional methodologies. For example, such
compounds can be made as described in U.S. Pat. No. 5,008,294.
[0148] Preparation of the NDGA Compounds
[0149] The NDGA Compounds and formulations thereof can be made by
any process conventional in the art. For example, the NDGA
Compounds can be made as described in, U.S. Pat. No. 5,008,294
(Jordan et al., issued Apr. 16, 1991); U.S. Pat. No. 6,291,524
(Huang et al., issued Sep. 18, 2001); Hwu, J. R. et al. (1998); or
McDonald, R. W. et al. (2001).
[0150] In one embodiment of the present invention, an NDGA
Compound, tetra-O-methyl NDGA, also known as
meso-1,4-bis(3,4-dimethoxyphenyl)-2,3-dimethylbutane, or M.sub.4N
is made as follows: a solution is made containing NDGA and
potassium hydroxide in methanol in a reaction flask. Dimethyl
sulfate is then added to the reaction flask and the reaction is
allowed to proceed. The reaction is finally quenched with water,
causing the product to precipitate. The precipitate is isolated by
filtration and dried in a vacuum oven. The compound is then
dissolved in a solution of methylene chloride and toluene and
subsequently purified through an alumina column. The solvents are
removed by rotary evaporation and the solid is resuspended in
isopropanol and isolated by filtration. The filter cake is dried in
a vacuum oven. The resulting tetra-O-methyl NDGA (M.sub.4N) is
crystallized by refluxing the filter cake in isopropanol and
re-isolating the crystals by filtration.
[0151] In some embodiments of the present invention, certain NDGA
Compounds of the present invention, such as G.sub.4N, also known as
meso-1,4-bis[3,4-(dimethylaminoacetoxy)phenyl]-(2R,3S)-dimethylbutane
or tetra-dimethylglycinyl NDGA, or a hydrochloride salt thereof and
similar compounds having amino acid substituents, can also be
prepared according to conventional methods, as described in, for
example, U.S. Pat. No. 6,417,234.
[0152] Compositions
[0153] The present invention further provides compositions,
including pharmaceutical compositions, comprising the catecholic
butanes including the NDGA Compounds and pharmaceutically
acceptable carriers or excipients. These compositions may include a
buffer, which is selected according to the desired use of the
catecholic butanes or NDGA Compounds, and may also include other
substances appropriate for the intended use. Those skilled in the
art can readily select an appropriate buffer, a wide variety of
which are known in the art, suitable for an intended use. In some
instances, the composition can comprise a pharmaceutically
acceptable excipient, a variety of which are known in the art.
Pharmaceutically acceptable excipients suitable for use herein are
described in a variety of publications, including, for example, A.
Gennaro (1995); Ansel, H. C. et al. (1999); and Kibbe, A. H.
(2000).
[0154] The compositions herein are formulated in accordance to the
mode of potential administration. Thus, if the composition is
intended to be administered intranasally or by inhalation, for
example, the composition may be a converted to a powder or aerosol
form, as conventional in the art, for such purposes. Other
formulations, such as for oral or parenteral delivery, are also
used as conventional in the art.
[0155] Compositions for administration herein may form solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders.
[0156] Therapeutic Methods
[0157] The catecholic butanes, including the NDGA Compound
compositions of the subject invention find use as therapeutic
agents in situations where one wishes to provide a treatment to a
subject who has a proliferative disease such as a malignant,
premalignant or benign tumor and where one wishes to provide
treatment to viral diseases such as HIV, HPV or HSV.
[0158] A variety of animal hosts are treatable according to the
subject methods, including human and non-human animals. Generally
such hosts are "mammals" or "mammalian," where these terms are used
broadly to describe organisms which are within the class mammalia,
including the orders carnivore (e.g., dogs and cats), rodentia
(e.g., guinea pigs, and rats), and other mammals, including cattle,
goats, horses, sheep, rabbits, pigs, and primates (e.g., humans,
chimpanzees, and monkeys). In many embodiments, the hosts will be
humans. Animal models are of interest for experimental
investigations, such as providing a model for treatment of human
disease. Further, the present invention is applicable to veterinary
care as well.
[0159] Moreover, the compounds of the present invention can be used
to treat a variety of tumors and cancers, including, without
limitation, acute lymphoblastic leukemia, acute myeloid leukemia,
adrenocortical carcinoma, anal cancer, astrocytoma, bile duct
cancer, bladder cancer, bone cancer osteosarcoma/malignant fibrous
histiocytoma, brain stem glioma, brain tumor ependymoma, brain
tumor medulloblastoma, breast cancer, carcinoid tumor
gastrointestinal, carcinoma adrenocortical, carcinoma islet cell,
cervical cancer, chronic lymphocytic leukemia, chronic myelogenous
leukemia, clear cell sarcoma of tendon sheaths, colon cancer,
cutaneous T-cell lymphoma, endometrial cancer, epithelial cancer
ovarian, esophageal cancer, Ewing's family of tumors, extragonadal
germ cell tumor, extrahepatic bile duct cancer, eye cancer,
intraocular melanoma, eye cancer retinoblastoma, gallbladder
cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor,
germ cell tumor extragonadal, germ cell tumor, ovarian tumor,
gestational trophoblastic tumor, glioma, hairy cell leukemia,
hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal
cancer, intraocular melanoma, islet cell carcinoma (endocrine
pancreas), Kaposi's sarcoma, laryngeal cancer, leukemia acute
lymphoblastic cancer, leukemia acute myeloid cancer, leukemia
chronic lymphocytic cancer, leukemia chronic myelogenous cancer,
leukemia hairy cell cancer, liver cancer, lung cancer non-small
cell, lung cancer small cell, male breast cancer, malignant
mesothelioma, medulloblastoma, melanoma, merkel cell carcinoma,
multiple endocrine neoplasia syndrome, mycosis fungoides, myeloma
multiple, nasal cavity, paranasal and sinus cancer, nasopharyngeal
cancer, neuroblastoma, oral cavity and lip cancer, oropharyngeal
cancer, osteosarcoma/malignant fibrous histiocytoma of bone,
ovarian epithelial cancer, ovarian germ cell tumor, pancreatic
cancer, parathyroid cancer, penile cancer, pheochromocytoma, pineal
and supratentorial primitive neuroectodermal tumors, pituitary
tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer,
renal, pelvis and ureter transitional cell cancer, retinoblastoma,
rhabdomyosarcoma, salivary gland cancer, sarcoma soft tissue adult,
Sezary syndrome, skin cancer, small intestine cancer, stomach
(gastric) cancer, testicular cancer, thymoma, thyroid cancer,
urethral cancer, uterine cancer endometrial, vaginal cancer, vulvar
cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.
[0160] Formulations, Dosages, and Routes of Administration
[0161] As mentioned above, an effective amount of the active agent
is administered to the host, where "effective amount" means a
dosage sufficient to produce a desired result. In some embodiments,
the desired result is at least a reduction or inhibition of tumor
growth as compared to a control.
[0162] Typically, the compositions of the instant invention will
contain from less than about 1% up to about 99% of the active
ingredient, that is, the catecholic butanes including the NDGA
Compounds herein; optionally, the instant invention will contain
about 5% to about 90% of the active ingredient. The appropriate
dose to be administered depends on the subject to be treated, such
as the general health of the subject, the age of the subject, the
state of the disease or condition, the weight of the subject, the
size of the tumor, for example. Generally, between about 0.1 mg and
about 500 mg or less may be administered to a child and between
about 0.1 mg and about 5 grams or less may be administered to an
adult. The active agent can be administered in a single or, more
typically, multiple doses. Preferred dosages for a given agent are
readily determinable by those of skill in the art by a variety of
means. Other effective dosages can be readily determined by one of
ordinary skill in the art through routine trials establishing dose
response curves. The amount of agent will, of course, vary
depending upon the particular agent used.
[0163] The frequency of administration of the active agent, as with
the doses, will be determined by the care giver based on age,
weight, disease status, health status and patient responsiveness.
Thus, the agents may be administered one or more times daily,
weekly, monthly or as appropriate as conventionally determined. The
agents may be administered intermittently, such as for a period of
days, weeks or months, then not again until some time has passed,
such as 3 or 6 months, and then administered again for a period of
days, weeks, or months.
[0164] The catecholic butanes or active agents of the present
invention can be incorporated into a variety of formulations for
therapeutic administration. More particularly, the catecholic
butanes of the present invention can be formulated into
pharmaceutical compositions by combination with appropriate,
pharmaceutically acceptable carriers or diluents, and may be
formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, aerosols,
liposomes, nanoparticles, granules, ointments, solutions,
suppositories, injections, inhalants and aerosols.
[0165] As such, administration of the active agents can be achieved
in various ways, such as oral, buccal, rectal, intranasal,
intravenous, intra-arterial, intra-tracheal, intraventricular,
intracranial, interstitial, transdermal, etc., or by inhalation or
implantation.
[0166] In particular, nanoparticle, micelle and liposomal
preparation can be administered systemically, including
parenterally and intranasally, as well as interstitially, orally,
topically, transdermally, via inhalation or implantation, such as
for drug targeting, enhancement of drug bioavailability and
protection of drug bioactivity and stability. Nanoparticle bound
drugs herein are expected to achieve prolonged drug retention in
tumors.
[0167] In pharmaceutical dosage forms, the active agents may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination, with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0168] For oral preparations, the active agents can be used alone
or in combination with appropriate additives to make tablets,
powders, granules or capsules, for example, with conventional
additives, such as lactose, mannitol, corn starch or potato starch;
with binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents. For oral rinses, the preparations can be made
in a manner conventional in the art, such as described in, for
example, Epstein, J. B. et al. (2002) and Pitten, F. et al.
(2003).
[0169] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are conventional in the
art. Suitable excipient vehicles are, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vehicle may contain minor amounts of
auxiliary substances such as pH adjusting and buffering agents,
tonicity adjusting agents, stabilizers, wetting agents or
emulsifying agents. Actual methods of preparing such dosage forms
are known, or will be apparent, to those skilled in the art. See,
e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 17th edition, 1985. The composition or formulation to
be administered will, in any event, contain a quantity of the agent
adequate to achieve the desired state in the subject being
treated.
[0170] The active agents can be formulated into preparations for
injection by dissolving, suspending or emulsifying them in an
aqueous or non-aqueous solvent, such as vegetable or other similar
oils, including corn oil, castor oil, synthetic aliphatic acid
glycerides, esters of higher aliphatic acids or propylene glycol;
and if desired, with conventional additives such as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers
and preservatives.
[0171] The active agents can be utilized in aerosol formulation to
be administered via inhalation. The compounds of the present
invention can be formulated into pressurized acceptable propellants
such as dichlorodifluoromethane, propane, nitrogen and the
like.
[0172] Furthermore, the active agents can be made into
suppositories by mixing with a variety of bases such as emulsifying
bases or water-soluble bases. The compounds of the present
invention can be administered rectally via a suppository. The
suppository can include vehicles such as cocoa butter, carbowaxes
and polyethylene glycols, which melt at body temperature, yet are
solidified at room temperature.
[0173] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier.
[0174] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0175] Kits with multiple or unit doses of the active agent, are
included in the present invention. In such kits, in addition to the
containers containing the multiple or unit doses of the
compositions containing the NDGA derivatives will be an
informational package insert with instructions describing the use
and attendant benefits of the drugs in treating pathological
condition of interest.
[0176] Tumors which may be treated using the methods of the instant
invention include carcinomas, e.g. colon, rectum, prostate, breast,
melanoma, ductal, endometrial, stomach, pancreatic, mesothelioma,
dysplastic oral mucosa, invasive oral tumor, non-small cell lung
carcinoma ("NSCL"), transitional and squamous cell urinary
carcinoma, etc.; neurological malignancies, e.g. neuroblastoma,
glioblastoma, astrocytoma, gliomas, etc.; hematological
malignancies, e.g. childhood acute leukaemia, non-Hodgkin's
lymphomas, chronic lymphocytic leukaemia, malignant cutaneous
T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma,
lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia,
bullous pemphigoid, discoid lupus erythematosus, lichen planus,
etc.; gynecological tumors, e.g., cervical and ovarian; testicular
tumors; liver tumors including hepatocellular carcinoma ("HCC") and
tumor of the biliary duct; multiple myelomas; tumors of the
esophageal tract; other lung tumors including small cell and clear
cell; Hodgkin's lymphomas; sarcomas in different organs; as well as
those mentioned above; and the like.
[0177] Preparation of Nanoparticles ("NP")
[0178] The present invention includes formulations of catecholic
butanes, including NDGA Compounds, in a NP preparation. A number of
different NP formulations suitable for use herein can be made
depending on the method of delivery. The NP formulation can differ
based on the drug release profile desired, by controlling the
molecular weight, the copolymer ratio, the drug loading, the
microparticle size and porosity and the fabrication conditions. The
NP formulations can also differ on the basis of polymers,
stabilizers, and surfactants used in the production process.
Different excipients may also have different effects on drug
uptake, drug distribution throughout the body and persistence of
the drug in plasma. A person having skills conventional in the art
will be able to determine the desired properties or
characteristics, and accordingly determine the appropriate NP
formulation to use.
[0179] The polymeric matrix of the NP must meet the criteria of
biocompatibility, bioavailability, mechanical strength and ease of
processing. The best known polymers for this purpose is the
biodegradable poly(lactide-co-glycolide)s ("PLGAs").
[0180] NP herein can be made by any process conventional in the
art. In one embodiment, the NP can be made as described in, for
example, Lockman, P. R., et al. (2002). The types of manufacturing
process include, for example, emulsion polymerization, interfacial
polymerization, desolvation evaporation and solvent deposition.
[0181] In the emulsion polymerization process of making the NP
herein, the polymerization process consists of building a chain of
polymers from a single monomer unit, as described in, for example,
Kreuter, J. (1994). Polymerization occurs spontaneously at room
temperature after initiation by either free radical or ion
formation, such as by use of high-energy radiation, UV light, or
hydroxyl ions. Once polymerization is complete the solution is
filtered and neutralized. The polymers form micelles and droplets
consisting of from about 100 to 10.sup.7 polymer molecules.
Surfactants and stabilizers are generally not need in this process.
Also, this process can be accomplished in an organic phase rather
than an aqueous phase.
[0182] The NP herein can also be made by an interfacial
polymerization process as described in, for example, Khouri, A. I.,
et al. (1986). In this process, monomers are used to create the
polymer and polymerization occurs when an aqueous and organic phase
are brought together by homogenization, emulsification, or
micro-fluidization under high-torque mechanical stirring. For
example, polyalkylcyanoacrylate nanocapsules containing the
catecholic butanes, such as the NDGA Compounds, can be made by
combining the lipophilic NDGA Compounds and the monomer in an
organic phase, dissolving the combination in oil, and slowly adding
the mixture through a small tube to an aqueous phase with constant
stirring. The monomer can then spontaneously form 200-300 nm
capsules by anionic polymerization. A variation of this process
involves adding a solvent mixture of benzyl benzoate, acetone, and
phospholipids to the organic phase containing the monomer and the
drug, as described in, for example, Fessi, H., et al. (1989). This
creates a formulation in which the drug is encapsulated and
protected against degradation until it reaches the target
tissue.
[0183] Macromolecules such as albumin and gelatin can be used in
oil denaturation and desolvation processes in the production of
NPs. In the oil emulsion denaturation process, large macromolecules
are trapped in an organic phase by homogenization. Once trapped,
the macromolecule is slowly introduced to an aqueous phase
undergoing constant stirring. The nanoparticles formed by the
introduction of the two immiscible phases can then be hardened by
crosslinking, such as with an aldehyde or by heat denaturation.
[0184] Alternatively, macromolecules can form NPs by "desolvation."
In the desolvation process, macromolecules are dissolved in a
solvent in which the macromolecules reside in a swollen, coiled
configuration. The swollen macromolecule is then induced to coil
tightly by changing the environment, such as pH, charge, or by use
of a desolvating agent such as ethanol. The macromolecule may then
be fixed and hardened by crosslinking to an aldehyde. The NDGA
Compounds can be adsorbed or bound to the macromolecule before
crosslinking such that the derivatives become entrapped in the
newly formed particle.
[0185] Solid lipid NP can be created by high-pressure
homogenization. Solid lipid NPs have the advantage that they can be
sterilized and autoclaved and possess a solid matrix that provides
a controlled release.
[0186] The present invention further includes NP with different
methods of drug loading. The NP can be solid colloidal NP with
homogeneous dispersion of the drug therein. The NP can be solid NP
with the drug associated on the exterior of the NP, such as by
adsorption. The NP can be a nanocapsule with the drug entrapped
therein. The NP can further be solid colloidal NP with homogeneous
dispersion of the drug therein together with a cell surface ligand
for targeting delivery to the appropriate tissue.
[0187] The size of the NPs may be relevant to their effectiveness
for a given mode of delivery. The NPs typically ranges from about
10 nm to about 1000 nm; optionally, the NPs can range from about 30
to about 800 nm; further typically, from about 60 to about 270 nm;
even further typically, from about 80 to about 260 nm; or from
about 90 to about 230 nm, or from about 100 to about 195. Several
factors influence the size of the NPs, all of which can be adjusted
by a person of ordinary skill in the art, such as, for example, pH
of the solution used during polymerization, amount of initiation
triggers (such as heat or radiation, etc.) and the concentration of
the monomer unit. Sizing of the NPs can be performed by photon
correlation spectroscopy using light scattering.
[0188] The NPs herein, such as polysaccharide NPs or albumin NPs,
may optionally be coated with a lipid coating. For example,
polysaccharide NPs can be crosslinked with phosphate (anionic) and
quarternary ammonium (cationic) ligands, with or without a lipid
bilayer, such as one containing dipalmitoyl phosphatidyl choline
and cholesterol coating. Other polymer/stabilizer include, but is
not limited to: soybean oil; maltodextrin; polybutylcyanoacrylate;
butylcayanoacrylate/dextran 70 kDa, Polysorbate-85;
polybutylcyanoacrylate/dextran 70 kDa, polysorbate-85; stearic
acid; poly-methylmethylacrylate.
[0189] The NP preparations containing the catecholic butanes, such
as the NDGA Compounds, such as by adsorption to the NPs, can be
administered intravenously for treatment of tumors, for example, in
the brain, heart and reticuloendothelial cell ("RES") containing
organs, such as liver, spleen and bone marrow. To avoid undesirable
uptake of these NP preparations by the reticuloendothelial cells,
the NPs may be coated with a surfactant or manufactured with a
magnetically responsive material.
[0190] Thus, optionally, a surfactant may be used in conjunction
with the NP. For example, polybutylcyanoacrylate NPs can be used
with a dextran-70,000 stabilizer and Polysorbate-80 as a
surfactant. Other surfactants include, but not limited to:
Polysorbate-20, 40, or 60; Poloxamer 188; lipid coating-dipalmitoyl
phosphatidylcholine; Epikuron 200; Poloxamer 338; Polaxamine 908;
Polaxamer 407. For example, Polyaxamine 908 may be used as a
surfactant to decrease uptake of NPs into the RES of the liver,
spleen, lungs, and bone marrow.
[0191] The magnetically responsive material can be magnetite
(Fe.sub.3O.sub.4) which can be incorporated into the composition
for making the NP. These magnetically responsive NPs can be
externally guided by a magnet.
[0192] In another embodiment, the NPs herein can be made as
described in Mu, L. and Feng, S. S. (2003), using a blend of
poly(lactide-co-glycolide)s ("PLGAs") and d-.alpha.-tocopheryl
polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS). The
latter can also act as an emulsifier, in addition to being a matrix
material.
[0193] Preparation of Micelle Forming Carriers
[0194] The present invention includes catecholic butanes, including
the NDGA Compounds, formulated in micelle forming carriers, where
the micelles are produced by processes conventional in the art.
Examples of such are described in, for example, Liggins, R. T. and
Burt, H. M. (2002); Zhang, X. et al. (1996); and Churchill, J. R.
and Hutchinson, F. G. (1988). In one such method,
polyether-polyester block copolymers, which are amphipathic
polymers having hydrophilic (polyether) and hydrophobic (polyester)
segments, are used as micelle forming carriers.
[0195] Another type of micelles is, for example, that formed by the
AB-type block copolymers having both hydrophilic and hydrophobic
segments, which are known to form micellar structures in aqueous
media due to their amphiphilic character, as described in, for
example, Tuzar, Z. and Kratochvil, P. (1976); and Wilhelm, M. et
al. (1991). These polymeric micelles are able to maintain
satisfactory aqueous stability irrespective of the high content of
hydrophobic drug incorporated within the micelle inner core. These
micelles, in the range of approximately <200 nm in size, are
effective in reducing non-selective RES scavenging and shows
enhanced permeability and retention at solid tumor sites. This
characteristic allows for the accumulation of anti-cancer drug,
such as the NDGA derivatives, to accumulate at the cancer site.
[0196] Further, for example,
poly(D,L-lactide)-b-methoxypolyethylene glycol (MePEG:PDLLA)
diblock copolymers can be made using MePEG 1900 and 5000. The
reaction can be allowed to proceed for 3 hr at 160.degree. C.,
using stannous octoate (0.25%) as a catalyst. However, a
temperature as low as 130.degree. C. can be used if the reaction is
allowed to proceed for about 6 hr, or a temperature as height as
190.degree. C. can be used if the reaction is carried out for only
about 2 hr.
[0197] In one embodiment, N-isopropylacrylamide ("IPAAm") (Kohjin,
Tokyo, Japan) and dimethylacrylamide ("DMAAm") (Wako Pure
Chemicals, Tokyo, Japan) can be used to make hydroxyl-terminated
poly(IPAAm-co-DMAAm) in a radical polymerization process, using the
method of Kohori, F. et al. (1998). The obtained copolymer can be
dissolved in cold water and filtered through two ultrafiltration
membranes with a 10,000 and 20,000 molecular weight cut-off. The
polymer solution is first filtered through a 20,000 molecular
weight cut-off membrane. Then the filtrate was filtered again
through a 10,000 molecular weight cut-off membrane. Three molecular
weight fractions can be obtained as a result, a low molecular
weight, a middle molecular weight, and a high molecular weight
fraction. A block copolymer can then be synthesized by a ring
opening polymerization of D,L-lactide from the terminal hydroxyl
group of the poly(IPAAm-co-DMAAm) of the middle molecular weight
fraction. The resulting poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide)
copolymer can be purified as described in Kohori, F., et al.
(1999).
[0198] The catecholic butanes, such as the NDGA Compounds, can be
loaded into the inner cores of micelles and the micelles prepared
simultaneously by a dialysis method. For example, a chloride salt
of the NDGA Compounds can be dissolved in N,N-dimethylacetamide
("DMAC") and added by triethylamine ("TEA"). The
poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) block copolymer can be
dissolved in DMAC, and distilled water can be added. The solution
of NDGA Compounds and the block copolymer solution can be mixed at
room temperature, followed by dialysis against distilled water
using a dialysis membrane with 12,000-14,000 molecular weight
cut-off (Spectra/Por.RTM.2, spectrum Medical Indus., CA. U.S.A.) at
25.degree. C. Poly(IPAAm-co-DMAAm)-b-poly(D,L-lactide) micelles
incorporating NDGA Compounds can be purified by filtration with a
20 nm pore sized microfiltration membrane (ANODISC.TM., Whatman
International), as described in Kohori, F., et al. (1999).
[0199] Preparation of Multivesicular Liposomes Containing NDGA
Compounds
[0200] Multivesicular liposomes ("MVL") can be produced by any
method conventional in the art, such as, for example, the double
emulsification process as described in Mantripragada, S. (2002).
Briefly, in the double emulsification process, a "water-in-oil"
emulsion is first made by dissolving amphipathic lipids, such as a
phospholipid containing at least one neutral lipid, such as a
triglyceride, in one or more volatile organic solvents, and adding
to this lipid component an immiscible first aqueous component and a
hydrophobic catecholic butane, such as a hydrophobic NDGA Compound.
The mixture is then emulsified to form a water-in-oil emulsion, and
then mixed with a second immiscible aqueous component followed by
mechanical mixing to form solvent spherules suspended in the second
aqueous component, forming a water-in-oil-in-water emulsion. The
solvent spherules will contain multiple aqueous droplets with the
catecholic butane, such as the NDGA Compound dissolved in them. The
organic solvent is then removed from the spherules, generally by
evaporation, by reduced pressure or by passing a stream of gas over
or through the suspension. When the solvent is completely removed,
the spherules become MVL, such as DepoFoam particles. When the
neutral lipid is omitted in this process, the conventional
multilamellar vesicles or unilamellar vesicles will be formed
instead of the MVL.
[0201] Formulation of Catecholic Butanes, Such as NDGA Compounds
for Oral Delivery
[0202] Some catecholic butanes, such as NDGA Compounds are
water-soluble, hydrophilic compounds, such as G.sub.4N. This
invention includes formulation of hydrophilic compounds in a
pharmaceutically acceptable carrier or excipient and delivery of
such as oral formulations, such as in the form of an aqueous liquid
solution of the compound, or the compounds can be lyophilized and
delivered as a powder, or made into a tablet, or the compounds can
be encapsulated.
[0203] The tablets herein can be enteric coated tablets. The
formulations herein can be sustained release, either slow release
or rapid release formulations.
[0204] The amount of the catecholic butanes, such as NDGA
Compounds, to be included in the oral formulations can be adjusted
depending on the desired dose to be administered to a subject. Such
an adjustment is within the skill of persons conventional in the
art.
[0205] Some catecholic butanes, including some NDGA Compounds, are
hydrophobic or lipophilic compounds, such as M.sub.4N. The
absorption of lipophilic compounds in the gut can be improved by
using pharmaceutically acceptable carriers that can enhance the
rate or extent of solubilization of the compound into the aqueous
intestinal fluid. Lipidic carriers are known in the art, such as,
for example, as described in Stuchlik, M. and Zak, S. (2001) The
formulations herein can be delivered as oral liquids or can be
encapsulated into various types of capsules.
[0206] The present invention includes, in one embodiment, a
formulation containing the lipophilic NDGA Compounds that are
formulated for oral delivery by dissolution of such compounds in
triacylglycerols, and the formulation is then encapsulated for oral
delivery. Triacyglycerols are molecules with long chain and/or
medium chain fatty acids linked to a glycerol molecule. The long
chain fatty acids range from about C.sub.14 to C.sub.24, and can be
found in common fat. The medium chain fatty acids range from about
C.sub.6 to C.sub.12, and can be found in coconut oil or palm kernel
oil. Triacylglycerols suitable for use herein include structured
lipids that contain mixtures of either short-chain or medium chain
fatty acids or both, esterified on the same glycerol molecule.
[0207] In another embodiment of the present invention, one or more
surfactants can be added to a mixture of catecholic butanes,
including NDGA Compounds, and lipidic carrier such that the drug is
present in fine droplets of oil/surfactant mix. The surfactants can
act to disperse the oily formulation on dilution in the
gastrointestinal fluid.
[0208] The present invention also includes a formulation for oral
delivery of the catecholic butanes, including NDGA Compounds, in
the form of a micro-emulsion consisting of hydrophilic surfactant
and oil. The micro-emulsion particles can be surfactant micelles
containing solubilized oil and drug.
[0209] Also suitable for oral administration are formulations of
the catecholic butanes, including NDGA Compounds, in a solid lipid
nanoparticle preparation. Solid lipid nanoparticles can be prepared
in any manner conventional in the art, such as, for example, as
described in Stuchlik, M. and Zak, S. (2001).
[0210] In one embodiment, the solid lipid nanoparticle can be
prepared in a hot homogenization process by homogenization of
melted lipids at elevated temperature. In this process, the solid
lipid is melted and the catecholic butane, such as the NDGA
Compound, is dissolved in the melted lipid. A pre-heated dispersion
medium is then mixed with the drug-loaded lipid melt, and the
combination is mixed with a homogenisator to form a coarse
pre-emulsion. High pressure homogenization is then performed at a
temperature above the lipids melting point to produce a
oil/water-nanoemulsion. The nanoemulsion is cooled down to room
temperature to form solid lipid nanoparticles.
[0211] In another embodiment of the present invention, the solid
lipid nanoparticles can be prepared in a cold homogenization
process. In this process, the lipid is melted and the catecholic
butane, such as the NDGA Compound, is dissolved in the melted
lipid. The drug-loaded lipid is then solidified in liquid nitrogen
or dry ice. The solid drug-lipid is ground in a powder mill to form
50-100 .mu.m particles. The lipid particles are then dispersed in
cold aqueous dispersion medium and homogenized at room temperature
or below to form solid lipid nanoparticles.
[0212] The present invention also includes formulation of the
lipophilic catecholic butanes, such as NDGA Compounds, in liposomes
or micelles for oral delivery. These formulations can be made in
any manner conventional in the art. Micelles are typically lipid
monolayer vesicles in which the hydrophobic drug associates with
the hydrophobic regions on the monolayer. Liposomes are typically
phospholipids bilayer vesicles. The lipophilic catecholic butane,
such as the lipophilic NDGA Compounds, will typically reside in the
center of these vesicles.
[0213] Intra-Arterial Administration
[0214] The present invention includes formulation of the catecholic
butanes, as exemplified by the NDGA Compounds, for intra-arterial
administration as is conventional in the art, as described in, for
example, Doolittle, N. D. et al. (2000); and Cloughesy, T. F. et
al. (1997), with or without accompanying blood brain barrier
disruption ("BBBD"), and with or without occlusion, such as in
hepatic artery chemoemobolization, as described in Drougas, J. G.
et al. (1998); and Desai, D. C. et al. (2001). Briefly, where NDGA
Compounds are administered intra-arterially with occlusion, primary
arteries leading to the target site are catheterized and the NDGA
Compounds are applied through a catheter. Embolization of the
arteries, in order to retain the NDGA Compounds at the target site
for a longer period, is performed using polyvinyl alcohol particles
alone or in combination with coils. Intra-arterial delivery of the
NDGA Compounds is limited to water soluble compositions. Water
soluble NDGA Compounds, such as G.sub.4N, for example, liposomal
formulations of hydrophobic NDGA Compounds, such as M.sub.4N, for
example, or nanoparticle formulations of hydrophobic NDGA Compounds
are particularly suited for this type of delivery. The drugs or
agents herein can be dissolved in saline prior to intra-arterial
injection and such injection may be preceded by heparin treatment
and sedation. For safest treatment of brain tumor, preferably,
intra-arterial administration is conducted before tumor burden
becomes excessive.
[0215] Osmotic disruption of the blood brain barrier ("BBB") as
conventional in the art may accompany intra-arterial delivery of
the agents herein as described in, for example, Doolittle, N. D. et
al. (2000); Sato, S. et al., Acta Neurochir (Wien) 140: 1135-1141;
disc 1141-1132 (1998); and Bhattacharjee, A. K. et al. Brain Res
Protocol 8: 126-131 (2001). Such a procedure can be used to
increase the transfer of drugs into the central nervous system
("CNS") preferably just prior to intra-arterial delivery. For such
disruption, a catheter is placed into an artery, usually the
superficial temporal artery, leading to the brain and the BBB is
disrupted with a solution of mannitol. This invasive procedure is
typically performed while the patient is under general anesthesia.
Such treatment may require prior hydration and administration of
anticonvulsants and/or atropine.
[0216] Formulation of NDGA Compounds for Intranasal Delivery
[0217] The present invention includes formulations of catecholic
butanes, as exemplified by the NDGA Compounds, for intranasal
delivery and intranasal delivery thereof. Intransal delivery may
advantageously build up a higher concentration of the active agents
in the brain than can be achieved by intravenous administration.
Also, this mode of delivery avoids the problem of first pass
metabolism in the liver and gut of the subject receiving the
drug.
[0218] The amount of the active agents that can be absorbed partly
depends on the solubility of the drug in the mucus, a composition
that consists of about 95% water solution of serum proteins,
glycoproteins, lipids and electrolytes. Generally, as lipophilicity
of the active agents herein increases, the drug concentration in
the CSF also increases. See, for example, Minn, A. et al.
(2002).
[0219] The hydrophilic NDGA Compounds can be dissolved in a
pharmaceutically acceptable carrier such as saline, phosphate
buffer, or phosphate buffered saline. In one embodiment, a 0.05 M
phosphate buffer at pH 7.4 can be used as the carrier, as described
in, for example, Kao, H. D., et al. (2000).
[0220] Intranasal delivery of the present agents may be optimized
by adjusting the position of the subject when administering the
agents. For example, the head of the patient may be variously
positioned upright-90.degree., supine-90.degree.,
supine-45.degree., or supine-70.degree., to obtain maximal
effect.
[0221] The carrier of the composition of NDGA Compounds may be any
material that is pharmaceutically acceptable and compatible with
the active agents of the composition. Where the carrier is a
liquid, it can be hypotonic or isotonic with nasal fluids and
within the pH of about 4.5 to about 7.5. Where the carrier is in
powdered form it is also within an acceptable pH range.
[0222] The carrier composition for intranasal delivery may
optionally contain lipophilic substances that may enhance
absorption of the active agents across the nasal membrane and into
the brain via the olfactory neural pathway. Examples of such
lipophilic substances include, but are not limited to, gangliosides
and phosphatidylserine. One or several lipophilic adjuvants may be
included in the composition, such as, in the form of micelles.
[0223] The pharmaceutical composition of active agents for
intranasal delivery to a subject for treatment of tumor and other
proliferative diseases, disorders, or conditions herein can be
formulated in the manner conventional in the art as described in,
for example, U.S. Pat. No. 6,180,603. For example, the composition
herein can be formulated as a powder, granules, solution, aerosol,
drops, nanoparticles, or liposomes. In addition to the active
agents, the composition may contain appropriate adjuvants, buffers,
preservatives, salts. Solutions such as nose drops may contain
anti-oxidants, buffers, and the like.
[0224] Delivery by Implantation
[0225] The catecholic butanes herein, as exemplified by the NDGA
Compounds, may be delivered to a subject for treatment by surgical
implantation into a tumor site, with or without surgical excision
of the tumor, such as by implantation of a biodegradable polymer
containing the NDGA Compounds. In one embodiment, this method of
treatment can be performed, for example, after surgical resection,
such as in the treatment and resection of brain tumor, as described
in, Fleming, A. B. and Saltzman, W. M., Pharmacokinetics of the
Carmustine Implant, Clin. Pharmacokinet, 41: 403-419 (2002). This
method of delivery is applicable to not only brain tumors but to
other tumors as well. This treatment may be combined with other
conventional therapy besides or in addition to surgery, such as
radiotherapy, chemotherapy or immunotherapy.
[0226] Thus, the biodegradable polymer herein can be any polymer or
copolymer that would dissolve in the interstitial fluid, without
any toxicity or adverse effect on host tissues. Preferably, the
polymer or monomers from which the polymer is synthesized is
approved by the Food and Drug Administration for administration
into humans. A copolymer having monomers of different dissolution
properties is preferred so as to control the dynamics of
degradation, such as increasing the proportion of one monomer over
the other to control rate of dissolution.
[0227] In one embodiment, the polymer is a copolymer of
1,3-bis-(p-carboxyphenoxy)propane and sebacic acid [p(CPP:SA)], as
described in Fleming A. B. and Saltzman, W. M., Pharmacokinetics of
the Carmustine Implant, Clin. Pharmacokinet, 41: 403-419 (2002);
and Brem, H. and Gabikian, P. (2001). In another embodiment, the
polymer is a copolymer of polyethylene glycol ("PEG") and sebacic
acid, as described in Fu, J. et al., (2002).
[0228] Polymer delivery systems are applicable to delivery of both
hydrophobic and hydrophilic NDGA Compounds herein. The NDGA
Compounds are combined with the biodegradable polymers and
surgically implanted at the tumor site. Some polymer compositions
are also usable for intravenous or inhalation therapy herein.
[0229] Delivery Through Inhalation
[0230] The catecholic butanes herein, as exemplified by the NDGA
Compounds, may be delivered systemically and/or locally by
administration to the lungs through inhalation. Inhalation delivery
of drugs has been well accepted as a method of achieving high drug
concentration in the pulmonary tissues without triggering
substantial systemic toxicity, as well as a method of accomplishing
systemic circulation of the drug. The techniques for producing such
formulations are conventional in the art. Efficacy against
pulmonary diseases may be seen with either hydrophobic or
hydrophilic NDGA Compounds delivered in this manner.
[0231] For pulmonary delivery via inhalation, the NDGA Compounds
herein may be formulated into dry powders, aqueous solutions,
liposomes, nanoparticles, or polymers and administered, for
example, as aerosols. Hydrophilic formulations may also be taken up
through the alveolar surfaces and into the bloodstream for systemic
applications.
[0232] In one embodiment, the polymers containing the active agents
herein are made and used as described in Fu, J. et al. (2002). For
example, the polymers herein can be polymers of sebacic acid and
polyethylene glycol ("PEG"), or can be poly(lactic-co-glycolic)
acid ("PLGA"), or polymers of polyethyleneimine ("PEI") and
poly-L-lysine ("PLL").
[0233] In another embodiment, the NDGA Compounds for inhalation
delivery may be dissolved in saline or ethanol before nebulization
and administered, as described in Choi, W. S. et al. (2001).
[0234] In a further embodiment, the agents herein are also
effective when delivered as a dry powder, prepared in the manner
conventional in the art, as described in, for example, Patton, J.
S. et al., Inhaled Insulin, Adv. Drug Deliv. Rev., 35: 235-247
(1999).
[0235] The present invention includes delivery of the NDGA
Compounds with the aid of microprocessors embedded into drug
delivery devices, such as, for example, SmartMist.TM. and AERx.TM.,
as described in, for example, Gonda, I., et al. (1998).
[0236] After reading the present disclosure, those skilled in the
art will recognize other disease states and/or symptoms which might
be treated and/or mitigated by the administration of formulations
of the present invention.
EXAMPLES
[0237] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric. Examples in the present tense
are prophetic examples.
Example 1
Preparation of a Preparative Batch of Tetra-O-Methyl-NDGA
[0238] Tetra-O-Methyl-NDGA, referenced herein as M.sub.4N, was
synthesized by the reaction of NDGA with excess dimethyl sulfate in
the presence of base, such as potassium hydroxide. The product was
isolated by the addition of water causing precipitation of the
product. The reaction product was passed through a plug of basic
alumina to remove traces of phenolic impurities, primarily various
species of di-O-methyl and tri-O-methyl-substituted NDGA. After the
solution of the reaction mixture had passed through the alumina
plug, the solvent was removed on a rotary evaporator giving a solid
product. This was triturated with 2-propanol, filtered and dried in
a vacuum oven to give crude tetra-O-methyl-NDGA. Crystallization
from 2-propanol gave tetra-.beta.-methyl-NDGA with a purity of
greater than or equal to 99.66%.
[0239] Step 1: Synthesis of Crude Preparation of
Tetra-O-Methyl-NDGA
[0240] A 22 L flask fitted with a mechanical stirrer, condenser and
inlet for inert atmosphere was set up in a tub for use as a cooling
bath. The flask was placed under an argon atmosphere, and was
charged with 484.3 grams of NDGA (Western Engineering &
Research Co, El Paso, Tex.), and 4850 mL of methanol and stirred.
To the stirred slurry was added a solution of 387.5 grams of
potassium hydroxide in 1210 mL of deionized water. The flask
containing this reaction mixture was cooled using an ice bath, and
dimethyl sulfate (1210 mL) was slowly added (dropwise). The
addition was controlled to avoid an exotherm. At the end of the
addition, the temperature was about 13.degree. C. The pH of the
reaction was monitored, and a 50% KOH solution was added in
portions during the day to maintain a basic pH; a total of 1400 mL
of 50% KOH solution was added. The reaction mixture with excess
base gave a pH of about 12, as detected using pH indicating strips.
The solution was dark at basic pH, but became light colored at
neutral or acidic pH.
[0241] At the end of the day, an additional 600 mL of dimethyl
sulfate was added, and the reaction mixture was allowed to stir
overnight. The next morning, the reaction was still basic, and the
reaction had progressed to about 90%.
[0242] The reaction mixture was quenched by the addition of 4850 mL
of deionized water, causing the product to precipitate. The product
was isolated by filtration, the filter cake washed thoroughly with
water, and the product dried in a vacuum oven at 50.degree. C. for
approximately 65 hr to give 539.5 g of the crude product. This
product was dissolved in 750 mL of methylene chloride, and to this
solution was added 375 mL of toluene. This solution was passed
through a short column of 2215 g of basic alumina. The alumina was
eluted with 12,000 mL of a methylene chloride/toluene solution
(2:1). Removal of the solvent in vacuo on a rotary evaporator gave
a solid residue. This was triturated with 1 L of 2-propanol. The
resulting slurry was filtered to isolate the solid product. This
was dried in a vacuum oven at 50.degree. C. under high vacuum for
approximately 21 hr to give 426.7 g (74% yield) of crude
tetra-O-methyl-NDGA.
[0243] Step 2--Crystallization of Tetra-O-Methyl-NDGA
[0244] A 3 L flask with mechanical stirrer, condenser, and inlet
was placed in a heating mantle, and was charged with 415.4 g of the
product. The flask was charged with 1245 mL of 2-propanol, and the
stirred mixture was heated to give a mild reflux; a solution was
obtained. The heat was turned off, and the mixture was allowed to
cool overnight. The crystalline product was isolated by filtration,
and the filter cake washed with 200 mL of cold 2-propanol. The
product was dried in a vacuum oven at 50.degree. C. under high
vacuum to constant weight giving 404.7 g (70.5% yield overall from
NDGA).
Example 2
Preparation of PLGA Nanoparticles Containing NDGA Compounds
[0245] The NDGA Compounds can be formulated as a nanoparticle
preparation in any manner conventional in the art. For example, the
nanoparticles can be prepared as described in Lamprecht, A. et al.
(2001a); and Lamprecht, A. et al. (2001b) and as follows.
[0246] The biodegradable polymer poly[DL-lactide-co-glycolide]
50/50 (PLGA) (mol. wt. 5,000 or 20,000) can be purchased from Wako
(Osaka, Japan). About 40 mg of a NDGA Compound can be dissolved in
4 ml of methylene chloride containing 250 mg of the polymer poly
[DL-lactide-coglycolide] 50/50 (mol. wt. 5,000 or 20,000). This
solution can thereafter be poured into 8 ml of aqueous polyvinyl
alcohol solution (1%) and homogenized with an ultrasonifier
(Ultrasonic Disruptor model UR-200P; Tomy Seiko Co., Ltd., Tokyo,
Japan) in an ice bath for 3 min. The methylene chloride can be
evaporated under reduced pressure, and the polymer precipitated.
The nanoparticles can be separated from the non-encapsulated drug
and free surfactant by centrifugation (14,000 g for 5 min).
Nanoparticles can be redispersed and centrifuged three times in
distilled water before lyophilization. Before oral administration,
the nanoparticles can be re-dispersed in phosphate buffer at pH
6.8.
[0247] The nanoparticles can be analyzed for their size
distribution and their surface potential using a Photal laser
particle analyzer LPA 3100 (Otsuka Electronics, Osaka, Japan) and a
Zetasizer II (Malvern Instruments, Worcestershire, U.K.)
respectively. The external morphology of the nanoparticles can be
analyzed with a JEOL JSM-T330A scanning microscope (Tokyo,
Japan).
Example 3
Preparation of PLGa/Vitamin E TPGS Nanoparticles with NDGA
Compounds
[0248] NPs containing PLGA and another matrix material,
d-a-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS
or TPGS), can be made as described in Mu, L. and Feng, S. S.
(2003), a modified oil-in-water single emulsion solvent
evaporation/extraction method. In this method, known amounts of
mass of polymer and NDGA Compounds are added into methylene
chloride (dichloromethane). The polymer, for example,
poly(DL-lactide-co-glycolide (PLGA; L/G=50/50, MW 40,000-75,000;
L/G=75/25, MW 90,000-120,000; and L/G=85/15, MW 90,000-120,000),
can be purchased from Sigma (USA). Vitamin E TPGS can be obtained
from Eastman Chemical, USA. The mixture is stirred to ensure that
all the materials are dissolved. The solution of organic phase is
then slowly poured in the stirred aqueous solution with or without
emulsifier and sonicated simultaneously at 50 W in pulse mode
(Misonix, USA). The formed o/w emulsion can be gently stirred at
room temperature (22.degree. C.) by a magnetic stirrer overnight to
evaporate the organic solvent. The resulting sample can be
collected by centrifugation, such as at 10,000 rpm, 10 min.
16.degree. C. (Eppendorf model 5810R, Eppendorf, Hamburg, Germany)
and washed once or twice with deionized water for some samples. The
produced suspension can be freezed dried (Alpha-2, Martin Christ
Freeze Dryers, Germany) to obtain a fine powder of nanoparticles,
which can be placed and kept in a vacuum dessicator.
Example 4
Preparation of Liposomes Containing NDGA Compounds
[0249] The NDGA Compounds, such as the lipophilic drugs, can be
encapsulated in long acting liposomes by processes conventional in
the art. One such method is described in, for example, Sharma, U.
S. et al. (1997).
[0250] Long-acting liposomes have extended blood circulation time.
They are typically composed of high phase-transition T.sub.m
lipids, high cholesterol content, and a component such as
phosphatidyl inositol, monosialoganglioside (GM.sub.1), or
synthetic phospholipids bearing a polyethylene glycol (PEG)
headgroup, which provides a steric barrier against plasma protein
access to the liposome surface.
[0251] In an example, liposomes composed of phosphatidylcholine
("PC"): cholesterol ("Chol"): polyethylene glycol conjugated to
dipalmitoylphosphatidylethanolamine ("PEG-DPPE") in a molar ratio
of 9:5:1 can be prepared. The lipids are initially mixed in
chloroform, and a thin film of lipid can be produced by evaporation
of the solvent. The lipids are then hydrated in a buffer consisting
of NaCl (145 mM), Tris[Hydroxymethyl]-2-aminoethane-sulfonic acid
(TES: 10 mM), and ethylenediamine tetraacetate (EDTA: 0.1 mM)
buffer, pH 7.2. The liposomes can then be extruded several times
through 0.08 .mu.m polycarbonate filters.
[0252] In another example, liposomes composed of
distearoylphosphatidylcholine ("DSPC): Chol: PEG-DSPE in at a molar
ratio of 9:5:1 can be prepared using a "remote loading" method as
described in Madden, T. D., et al. (1990). This remote loading
method allows for encapsulation of high concentration of NDGA
Compounds within the liposome aqueous core. Briefly, a thin film of
lipids can be hydrated in ammonium sulfate (250 mM, pH 5.5). The
lipid suspension can be extruded through 0.08 .mu.m polycarbonate
filters at 60.degree. C. and dialyzed overnight against isotonic
sucrose to remove free ammonium sulfate. Hydrophilic NDGA Compounds
can be hydrated in 10% (w/v) sucrose and incubated with the
preformed liposomes for 1 hr at 65.degree. C. The preparation can
be dialyzed against isotonic sucrose to remove the minor residual
fraction of unencapsulated drug. This method may yield
encapsulation efficiencies of greater than or equal to 90% of the
initial NDGA compounds.
[0253] Poly(lactide-co-glycolide)-monomethoxy-poly(polyethylene
glycol) (PLGA-mPEG) copolymers of different molar ratios can be
prepared by a melt polymerization process under vacuum using
stannous octoate as catalyst, as described in Beletsi, A et al.
(1999); and Avgoustakis, K. et al. (2002).
Example 5
Preparation of Intranasal Formulations of NDGA Compounds
[0254] The NDGA Compounds can be formulated as a dry powder or an
aerosol for intranasal delivery by any methods conventional in the
art, such as, for example, as described in Marttin, E. et al.
(1997).
[0255] In one embodiment, the NDGA Compound is formulated as a
solution with randomly methylated .beta.-cyclodextrin ("RAMEB")
(degree of substitution 1.8) (Wacker, Burghausen, Germany),
mannitol or glucose in MQ water, water that is filtered by a Mili-Q
UF plus ultrapure water system from Millipore (Etten-Leur, The
Netherlands). This formulation may be administered as a spray or as
drops. The dose of NDGA Compound in the liquid formulation may be
from about 1 mg/ml to about 1500 mg/ml, or optionally from about 10
mg/ml to about 1200 mg/ml, or further optionally from about 100
mg/ml to about 1000 mg/ml, or still optionally, from about 200
mg/ml to about 800 mg/ml, or any value that falls between these
ranges. These liquid formulations can be sprayed into the nostril
or applied as drops.
[0256] In another embodiment, the present invention includes
lyophilized powder formulations of NDGA Compounds, prepared by
dissolving the NDGA Compounds and various amounts of RAMEB,
lactose, or mannitol in MQ water, and lyophilizing the mixture,
such as, for example, overnight.
Example 6
Production of a Biodegradable Polymer Implant
[0257] The NDGA Compounds herein can be incorporated into a
biodegradable polymer for implantation into tumors that are not
operable. Such biodegradable polymer can be made by any method
conventional in the art, such as described in Fleming, A. B. and
Saltzman, W. M. (2002). Typically, the polymer implant is inserted
after removal of the bulk of the tumor. One or more wafers of this
biodegradable polymer can be implanted at one time depending on the
dose of the compounds desired. The biodegradable matrix of the
polymer can be made up of polifeprosan 20, a copolymer of
1,3-bis-(p-carboxyphenoxy)propane and sebacic acid [p(CCP:SA)] in a
20:80 molar ratio. To form the polymer for implant, p(CPP:SA) and a
compound herein can be co-dissolved in dichloromethane and spray
dried to form spherical particles with a size range of about 1 to
about 20 .mu.m. The resulting "microspheres" are compression
moulded to form wafers of any desired size, such as, for example,
about 14 mm in diameter and about 1 mm in thickness. The wafers
have a homogeneous structure consisting of densely packed
microspheres surrounded by small gaps. Concentration of the NDGA
Compounds can be in any amount appropriate for the subject to be
treated, such as, for example, 3.8% active compound.
Example 7
Preparation of PLGA-mPEG Nanoparticles
[0258] PLGA-mPEG nanoparticles containing the NDGA Compounds can be
prepared using the double emulsion method described by Song C. X.
et al (1997), with minor modifications. Here, an aqueous solution
of the NDGA Compounds can be emulsified in dichloromethane in which
the copolymer is dissolved, using probe sonication (Bioblock
Scientific, model 75038). This water/oil emulsion can be
transferred to an aqueous solution of sodium cholate and the
mixture can be probe sonicated. The resulting water/oil/water
emulsion formed can be gently stirred at room temperature until
evaporation of the organic phase is complete. The nanoparticles
made in this way can be purified by centrifugation and
reconstituted with deionized and distilled water. The nanoparticles
can then be filtered such as through a 1.2-.mu.m filter (Millex A
P, Millipore).
Example 8
Preparation of Pluronic Micelles Containing NDGA Compounds
[0259] Pluronic is a triblock PEO-PPO-PEO copolymer, with PEO
representing poly(ethylene oxide), and PPO representing
polypropylene oxide). The hydrophobic central PPO blocks form
micelle cores, while the flanking PEO blocks form the shell or
corona, which protects the micelles from recognition by the
reticuloendothelial system ("RES"). Pluronic copolymers are
commercially available from BASF Corp, and ICI. The NDGA Compounds
can be introduced into the Pluronic micelles by any method
conventional in the art, as described in, for example, Rapoport, N.
Y., et al. (1999).
[0260] Briefly, the NDGA Compounds can be dissolved in PBS or RPMI
medium, followed by a short, such as 15 sec, sonication in a
sonication bath operating at 67 kHz. The solution can be kept for
about 2 hr at 37.degree. C., upon which the non-solubilized drug
can be removed by dialysis through a 1000 D cutoff membrane at
37.degree. C. for about 12 hr against PBS or RPMI medium (dialysis
to be done only for 10 and 20 wt % Pluronic solutions).
Example 9
Administration of NDGA Compounds by Implantation
[0261] Implantation of the NDGA Compounds herein can be done in any
manner conventional in the art. In one embodiment, implantation is
performed as described in Brem, H., and Gabikian, P. (2001). It is
preferable that prior to the insertion of the polymer implant that
the tumor be surgically debulked. Further the dura should be closed
in a water-tight fashion to eliminate cerebrospinal fluid leakage
and to decrease risk of infection. It is also desirable to use
preoperative anti-convulsants and high dose steroids as necessary
for neurologic compromise. It is further desirable to continue
steroid therapy for at least 2 weeks post-operatively.
Example 10
Delivery of NDGA Compounds in ethanol Via Inhalation
[0262] The NDGA Compounds herein can be delivered via inhalation
using any formulation conventional in the art, including as dry
powders or as aqueous solutions. The former has the advantage of
stability, low susceptibility to microbial growth and high mass per
puff. Aqueous solutions offer better reproducibility and avoid the
issue of clumping.
[0263] In one embodiment, certain of the NDGA Compounds are
delivered according to the method as described in Choi, W. S. et
al. (2001). Depending on the particular compound and the solubility
thereof, the compounds can be formulated to an appropriate
concentration in ethanol, such as, for example in a range of from
about 1 mg/ml to about 1000 mg/ml, or any intervening values
in-between, such as, for example, between about 2 mg/ml and about
800 mg/ml, or between about 4 mg/ml and about 100 mg/ml, or between
about 5 mg/ml and about 50 mg/ml. Aerosol particles of 1-3 .mu.m
size can be generated for maximal deep lung delivery. For better
solubility of the compounds in ethanol, the compounds herein can be
first lyophilized, then acidified if necessary or desirable, such
as with H.sub.3PO.sub.4. The pH of the resulting composition can be
adjusted with NaOH, if desired, such as to pH 7.4. The resulting
composition can then be lyophilized, suspended in ethanol,
sonicated and stirred to produce appropriate submicron size
particles. The aerosolized compounds can then be administered using
a standard commercial nebulizer, such as a compressor (air jet) or
an ultrasonic type, or a metered dose inhaler. An example is a PARI
LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, Calif.) in
conjunction with a PARI PRONEB compressor. A volume of about 9 ml
can be charged in the reservoir of the nebulizer and nebulized for
up to about 10 min.
[0264] In another embodiment, the formulation for inhalation can be
prepared as described in Wang, D. L., et al. (2000). For example,
powdered NDGA Compounds can be dissolved in 10:90 (v/v)
polyethylene glycol 300:100% ethanol containing 0.5% (w/v) ascorbic
acid and 0.5% (w/v) phosphatidylcholine. The drug formulation can
then be aerosolized using a Pari LC-plus nebulizer (Pari, Richmond,
Va.) and a subject to be treated can be exposed to the aerosol
generated for varying lengths of time, depending on the dose of the
formulation and the desired concentration to be achieved. Such
periods of time can be about 5 minutes, 10 minutes, 15 minutes or
longer.
Example 11
Delivery of NDGA Compounds Using Specially Designed Inhalator
[0265] The NDGA Compounds can also be formulated in a number of
other pharmaceutically acceptable carriers for inhalation purposes.
In this example, certain of the compounds herein can be delivered
according to the method of Enk, A. H. et al. (2000). Such compounds
can be dissolved in a solution containing about 5% glucose and 2%
human albumin Inhalation can then be performed using a specially
designed inhalator. (Jetair, Fa. Hoyer, Germany).
Example 12
Delivery of NDGA Derivatives as an Oral Rinse for Treatment of Oral
Lesions
[0266] The delivery of NDGA derivatives to the oral cavity involves
the use of an oral rinse using excipients that are conventional in
the art, such as, for example, that described in Armstrong W. B.,
et al. (2000). The NDGA derivatives are dispensed as a powder that
is reconstituted in an appropriate delivery fluid, such as Roxane
Saliva Substitute (Roxane Laboratories, Columbus, Ohio),
immediately before use. Patients then hold the NDGA derivative
suspension in the mouth for about 1 minute before expectorating or
swallowing the drug mixture. This procedure is carried out at least
once daily for local delivery of NDGA derivatives to the oral
cavity.
[0267] Alternatively, the delivery of NDGA derivatives to the oral
cavity can involve an oral rinse formulation such as described in
Epstein, J. B., et al. (2001). Briefly, the NDGA derivatives are
prepared in an oral rinse containing about 0.1% alcohol and
sorbitol. Patients are provided with a suitable volume, such as
about 5 ml of the rinse, to be rinsed in the mouth for about 1
minute and expectorated. This procedure is carried out at least
once daily for local delivery of NDGA derivatives to the oral
cavity.
Example 13
Arrest of Tumor Growth in Mice After Systemic or Oral
Administration of M.sub.4N
[0268] In this example, the inventors considerably expanded the
cancer therapeutic potential of M.sub.4N by investigating both its
anti-tumor efficacy in vivo against several human cancer xenograft
models, and its ability to be administered systemically through
various routes of administration at pharmacologically relevant
levels. This example shows the following: (1) when administered in
vivo through differing routes of systemic administration including
intraperitoneal (IP) injection, intravenous (IV) injection, and
oral feeding, M.sub.4N distributes consistently to various organs
and to tumors with little or no apparent toxicity to mice; (2)
systemic IP administration of M.sub.4N effectively retards the in
vivo growth of xenografts from 4 human cancer cell types: MCF-7
breast adenocarcinoma, Hep3B hepatocellular carcinoma, HT-29
colorectal carcinoma, and LNCaP prostate carcinoma; and systemic
oral administration of M.sub.4N effectively suppresses the growth
of LNCaP xenograft tumors--only LNCaP tumors have thus far been
assessed in oral administration efficacy studies.
[0269] Cell Lines and Culture Conditions. Human tumor cell lines
were purchased from ATCC (Mannassas, Va.). The human hepatocellular
carcinoma cell line, Hep 3B, and the human breast epithelial
adenocarcinoma cell line, MCF-7, were grown in Eagle's Minimal
Essential Media +10% FBS+penicillin+streptomycin. The human
colorectal adenocarcinoma cell line, HT-29, was grown in McCoy's 5a
Medium+10% FBS+penicillin+streptomycin. The human prostate
carcinoma cell line, LNCaP was grown in RPMI 1640+10%
FBS+penicillin+streptomycin.
[0270] Mice. Female ICR mice, 6-8 weeks of age, were purchased from
Harlan Sprague Dawley (Indianapolis, Ind.). C57bl/6 mice were
purchased from Charles River Laboratories (Wilmington, Mass.).
Athymic (thy.sup.-/thy.sup.-) nude mice, males and females 5-6
weeks of age, were purchased from Charles River Laboratories and
were housed in a pathogen-free room under controlled temperature
and humidity in accordance with Institutional Animal Care and Use
Guidelines. C57bl/6 mice bearing C3 cell-induced tumors were
prepared as described in Kim, E. H. et al. (2004).
[0271] Xenograft Assay of Human Tumors. Athymic nude mice were
implanted subcutaneously in their flanks with 2.5.times.10.sup.6
Hep3B cells, 2.times.10.sup.6 LNCaP cells, 1.times.10.sup.7 HT-29
cells, or 2.times.10.sup.6 MCF-7 cells. After the tumors exhibited
a mean diameter of 7-8 mm, the mice were assigned to one of two
groups: a control group receiving vehicle only, and a group
receiving M.sub.4N dissolved in the Cremaphor EL-ethanol-based
solvent system. Assignment was made so that both the control group
and the experimental group contained mice bearing tumors of
comparable sizes.
[0272] M.sub.4N was dissolved in 6% Cremaphor EL, 6% ethanol, 88%
saline as described in Loganzo et al. (2003). Mice received a
single daily 100 .mu.L i.p. injection containing 2 mg of M.sub.4N
for 3 weeks. The control mice received an equal volume of the
vehicle. Tumors were measured in two perpendicular dimensions (L
and W) once every seven days, and the tumor volumes were calculated
according to the following formula:
V=(L.times.W/2).sup.3.times..pi./6. The results from the individual
mice were plotted as average tumor volume versus time. Statistical
significance of the mean differences in tumor volume was assessed
by Student's t-test. At the termination of the experiment, tumor
biopsies were collected for immunohistological analysis of cdc2 and
survivin expression.
[0273] M.sub.4N Tissue Distribution Studies Using .sup.3H-M.sub.4N.
Harlan ICR mice or C3 cell-induced tumor-bearing C57bl/6 mice were
injected via tail vein or intraperitoneally with 100 .mu.L of
Cremaphor-ethanol based solvent containing 100 .mu.Ci of tritiated
M.sub.4N and 60 mM of cold M.sub.4N. At the specified time
post-injection, the mice were sacrificed, the organs and blood were
collected, weighed, then dissolved overnight in 4 M guanidine
isothiocyanate (GITC). The insoluble pellet was then further
extracted with EtOH. Tritium content of both the GITC extract and
the EtOH extract was measured on a Packard scintillation counter
and the quantity of M.sub.4N in each organ was calculated based on
the specific activity of the inoculum.
[0274] Tissue Distribution and Toxicity Analysis Following
Short-Term and Long-Term Oral Feeding. For short-term feeding
experiments, 30 mg of M.sub.4N dissolved in 300 .mu.L castor oil
was orally administered to each of 6 mice. At 2 h, 4 h, and 8 h
post-administration time points, 2 mice were sacrificed, the organs
and blood were collected, and the M.sub.4N extracted and
quantitated as described below. In long-term feeding experiments,
mice were fed food balls consisting of M.sub.4N dissolved in corn
oil and Basal Mix (Harlan Teklad; Madison, Wis.; Cat. # TD 02273)
for 14 weeks. Food balls weighed 9 g and contained 242 mg M.sub.4N
each. Two mice, one male and one female, were reserved for
long-term drug retention studies; and fourteen mice, both male and
female, were used for long-term drug toxicity studies. At the end
of feeding, mice were sacrificed, the organs and blood were
collected, and the M.sub.4N extracted and quantitated as described
below.
[0275] M.sub.4N Extraction and HPLC Analysis Following Oral
Feeding. Organs and blood were harvested from M.sub.4N-fed mice,
then frozen overnight at -80.degree. C. Prior to freezing,
gastro-intestinal organs (stomach, small intestine, colon) were cut
open longitudinally and washed thoroughly with PBS to remove any
contents. The following day, organs were cut into small pieces on
dry ice with a razor blade, dried in a Speed-vac, then crushed into
a rough powder using a mortar and pestle. Samples were extracted
overnight in 100% ethanol with shaking Samples were centrifuged and
the supernatant collected. Pellets were extracted two more times in
100% ethanol overnight with shaking. The pooled ethanol extracts
were evaporated on bench top for several days, then re-extracted
with ethyl acetate, and dried completely in a Speed-vac. The dried
samples were then analyzed quantitatively by HPLC and M.sub.4N was
identified by mass spectroscopy using pure M.sub.4N as a
standard.
[0276] In samples from short-term fed mice, dialysis was performed
to further purify M.sub.4N from the tissue extracts. Dried ethanol
extracts were redissolved in 1.5 mL 100% EtOH and centrifuged for 5
min. The supernatant was collected and the pellet was resuspended
in 0.4 mL ethanol and centrifuged again. The supernatant was pooled
with the previous supernatant, then dialyzed overnight against 150
mL 100% EtOH. The dialysates were dried on bench top and in a
speed-vac, then analyzed by HPLC.
[0277] HPLC Quantitation of M.sub.4N. Samples from a single mouse
at each time point were sent to KP Pharmaceuticals (Bloomington,
Ind.) for HPLC analysis. HPLC conditions were described as follows:
35%:0.1% TFA in H.sub.2O, 65%="CAN." The M.sub.4N standard was
prepared by diluting 10.01 mg M.sub.4N in 100 mL of CAN, then
sonicating for 5 min. (2002 ng/injection). The samples were
prepared by adding 400 .mu.L EtOH and sonicating for 2 min. or
until complete dissolution was achieved. The injection volume for
the samples was 100 .mu.L.
[0278] M.sub.4N is Distributed Systemically to Various Tissues and
With No Detectable Toxicity Following Intraperitoneal, Intravenous,
and Oral Administration
[0279] Systemic Distribution of M.sub.4N Following a Single
Intraperitoneal or Intravenous Administration
[0280] Previous studies demonstrated substantial tumoricidal
activity following localized intratumoral injection of M.sub.4N
into C3 cell-induced tumors in mice, as described in U.S. Pat. No.
6,608,108. Yet, with few exceptions, the clinical use of
nonsystemic intratumoral chemotherapy is rare even for high
mortality cancers characterized by well defined primary lesions
i.e. breast, colorectal, lung, and prostate. Rather, the
conventional wisdom and standard of care in clinical oncology
remains surgery followed by systemic chemotherapy and/or radiation
as deemed appropriate to the clinical situation. Because the
effective treatment of many primary tumors as well as metastatic
disease requires systemic delivery, the ability to distribute
M.sub.4N systemically in vivo was assessed.
[0281] A mixture of tritiated and cold M.sub.4N was dissolved in a
6% Cremaphor EL, 6% ethanol, 88% saline solvent then injected
intraperitoneally (i.p.) and intravenously (i.v.) via tail vein
into mice. At 3 hours post-injection, the organs and blood were
harvested and weighed, and the M.sub.4N was extracted. The tritium
content of the extracts from each organ was measured on a Packard
scintillation counter and the quantity of M.sub.4N in each organ
was calculated based on the specific activity of the inoculum. As
shown in FIGS. 1A and 1B, M.sub.4N was successfully distributed to
various organs at 3 hours post-injection by both i.p. and i.v.
routes of administration. Interestingly, very similar profiles of
tissue distribution were obtained despite the different routes of
administration, thus indicating a non-random, perhaps regulated
mechanism of drug dispersal. Corroborating this, a very similar
profile of distribution was observed using an oral route of
administration described below. The majority of the recovered
radioactivity localized to the gastrointestinal tract organs (FIG.
1A): the stomach, small intestine, caecum, and large intestine, in
the range of 3 .mu.g to 20 .mu.g of M.sub.4N per gram of tissue.
Significant quantities of M.sub.4N were also present in the liver
and fat, and lower concentrations in the range of 150 to 400 ng per
gram tissue (FIG. 1B) were detected in the brain, kidneys and
spleen. Little or no M.sub.4N, however, was detected in the heart
or the blood at 3 hours post-injection. In conclusion, M.sub.4N may
be safely and systemically administered to various specific tissues
via i.p. or i.v. injection.
[0282] The previous experiment demonstrated that M.sub.4N may be
delivered systemically and relatively rapidly to various tissues at
a single time point. To determine the distribution of M.sub.4N in
tissues over time following a single application, and also to
assess the ability to deliver M.sub.4N to a distant tumor, six C3
cell-induced tumor bearing mice (A-F) were treated i.p. with
.sup.3H-- M.sub.4N as described above. At 4 hours, 6 hours, 18
hours, and 6 days post-injection, the quantities of .sup.3H--
M.sub.4N in various tissues and the tumors were measured. The
results shown in FIG. 2 confirmed the ability to distribute
M.sub.4N systemically, with the majority of M.sub.4N again
localizing to the GI tract organs, fat and liver, and lesser
amounts detected in the brain and kidneys. Interestingly, and not
apparent in the previous 3 hour injection, the fat and spleen
exhibit a rapid increase in M.sub.4N levels between 4 hours and 6
hours. A significant, although relatively low amount of M.sub.4N,
294 ng M.sub.4N per gram of wet tumor, was measured in the tumor at
6 hours post-injection. The changes in tissue distribution of
M.sub.4N following initial application show an increase in M.sub.4N
levels in these tissues from 0 to 6 hours with a peak occurring at
approximately 6 hours. At 18 hours, M.sub.4N levels had
substantially decreased, and at 6 days post-injection, although
significant M.sub.4N levels could still be detected in most
tissues, M.sub.4N levels had decreased to 5-10% of levels seen at 6
hours.
[0283] Systemic Tissue Distribution Following Short-Term and
Long-Term Oral Feeding and In Vivo Toxicity Evaluations.
[0284] The previous experiments demonstrated that M.sub.4N can be
systemically distributed in vivo by i.p. and i.v. injection with no
apparent toxicity. The convenience and ease of oral administration,
however, especially in the case of long-term post-surgical adjuvant
treatment, would considerably facilitate drug administration to
patients and would improve patient quality of life. Thus, in
addition to i.p. and i.v. administration, the ability to
systemically distribute M.sub.4N by oral administration was also
investigated. In both short-term (<8 hours) feeding experiments
and long-term (14 weeks) feeding experiments, M.sub.4N levels in
various tissues and their in vivo toxicity was assessed. In
short-term experiments, mice were fed 30 mg of M.sub.4N dissolved
in castor oil (100 mg M.sub.4N/mL castor oil), and at 2, 4, and 8
hours post-feeding, the quantity of M.sub.4N present in various
tissues was determined by HPLC. As shown in Table 1, a relatively
very low quantity of M.sub.4N (<2 ng per gram tissue) was found
in each tissue at 2 hours post-feeding. Between 2 and 4 hours
post-feeding, most organs including the liver, pancreas, kidneys,
seminal vesicles, small intestine, stomach, large intestine,
caecum, and blood exhibited a large increase in M.sub.4N levels. At
4 hours, as was seen in the i.p. and i.v. administrations, most of
the M.sub.4N localized to the gastro-intestinal tract organs, in
the range of 4 ng to 45 ng of M.sub.4N per gram of tissue.
Significant quantities of M.sub.4N were also present in the
pancreas, and lower concentrations in the range of 0.1 ng to 2 ng
per gram tissue were detected in the heart, liver, seminal
vesicles, blood, and bladder. At 8 hours post-feeding, M.sub.4N
levels had decreased in nearly all organs, and most of the organs
had been cleared of M.sub.4N. In conclusion, M.sub.4N was
distributed transiently to various organs following a single oral
administration of 30 mg of M.sub.4N. M.sub.4N levels peaked at
roughly 4 hours post-feeding, and M.sub.4N concentrations were
significantly lower than seen in i.p. and i.v. single
administrations.
TABLE-US-00001 TABLE 1 M.sub.4N (ng)/organ dry weight (g) M.sub.4N
(.mu.g/g) Organ 2 hours 4 hours 8 hours 14 weeks Heart 0.83 0.11
1.16 12.62 Liver 0.05 0.52 0 5.89 Lungs 0.43 0 2.7 23.09 Pancreas
0.57 24.69 2.97 28.81 Kidneys 0.12 0.61 0 8.33 Seminal Vesicles
0.21 0.37 0 16.79 Small Intestine 0.09 8.42 5.53 906.27 Stomach
0.23 45.03 33.9 409.27 Lg. Intestine 0.24 8.24 2.68 350.88 Caecum
1.11 4.38 6.55 440.23 Spleen 1.1 0.16 0 302.78 Blood 0.5 2.08 0
23.13 Bladder 2.34 1.69 0 6.24 Fat 0.11 0 0 17.41
[0285] The objective of the long-term feeding experiments was to
measure the steady state levels of M.sub.4N in various mouse organs
following continuous oral administration for 14 weeks. Food balls
weighing approximately 9 g and containing approximately 280 mg
M.sub.4N were continually fed to wild-type mice for 14 weeks. A
single 9 g food ball is consumed by a single mouse in about 3 days,
which translates to 93.3 mg of M.sub.4N consumed or administered
daily. HPLC quantitation showed that oral administration had
systemically distributed M.sub.4N to all organs analyzed; and
surprisingly had accumulated in all organs to concentrations
greatly exceeding those seen previously for i.p., i.v., and oral
one time administrations. Between 350 .mu.g and 900 .mu.g M.sub.4N
per gram tissue was measured in the GI tract organs and the spleen;
15 .mu.g/g to 30 .mu.g/g M.sub.4N was measured in the lungs,
pancreas, seminal vesicles, blood, and fat; and 5 .mu.g/g to 13
.mu.g/g M.sub.4N was measured in the heart, liver, kidneys, and
bladder.
[0286] Despite the high levels of M.sub.4N present in various
organs following systemic long-term oral administration, no signs
of toxicity were seen as determined by daily evaluation of activity
and overall body weight change (FIG. 3).
[0287] Systemic M.sub.4N treatment inhibits the in vivo growth of
human tumor xenografts.
[0288] Based on (1) our cell culture analyses showing that M.sub.4N
will effectively prevent the growth of various human tumor cells,
and (2) our in vivo observations that M.sub.4N can be distributed
systemically at non-toxic doses, we investigated whether systemic
administration of M.sub.4N would inhibit the in vivo growth of
human tumors. Athymic nude mice were implanted s.c. in each flank
with MCF-7 breast adenocarcinoma cells, Hep3B hepatocellular
carcinoma cells, HT-29 colorectal carcinoma cells, and LNCaP
prostate carcinoma cells. Most mice developed tumors in both
flanks, although some developed a single tumor. When tumors
attained a mean diameter of 7-8 mm, mice received for three weeks a
single daily i.p. injection containing 2 mg of M.sub.4N dissolved
in 100 uL Cremaphor-ethanol based solvent. Control mice received
vehicle only. Tumors were measured in two perpendicular dimensions
(L and W) once every seven days, and tumor volumes were calculated
according to the formula: V=(L.times.W/2).sup.3.times..pi./6.
[0289] As shown in FIG. 4, systemic M.sub.4N treatment for 21 days
resulted in statistically significant (p<0.05) reductions in
mean tumor growth in all four tumor types. After 21 days of
systemic M.sub.4N treatment, MCF-7 tumors were reduced 74% in mean
tumor volume to being only 25.5% of the mean volume of the control
tumors (Table 2); HT-29 tumors were reduced 70% in mean volume; Hep
3B liver tumors were reduced 80%; and LNCaP prostate tumors were
reduced 53%.
TABLE-US-00002 TABLE 2 Tumor Volume Increase (%) - 21 Days
Treatment Tumor M.sub.4N- Ratio of Mean M.sub.4N-Treated Tumor Size
Type Treatment Control to Control Tumor Size McF-7 84% 624% 25.5%
Ht-29 787% 2920% 29.4% Hep 3B 113% 1001% 19.3% LNCaP 25% 171%
46.3%
[0290] Table 3 shows the total number of tumors in each treatment
group, and categorizes each based on whether there was an overall
increase or decrease in size over the 21 days of treatment. In all
four tumor types, 100% of the control tumors each exhibited an
increase in tumor size. However, among the M.sub.4N-treated mice, 7
out of 10 MCF-7 tumors decreased in size; 2 out of 7 Hep3B tumors
decreased in size, 3 out of 11 HT-29 tumors decreased in size, and
9 out of 11 LNCaP tumors decreased in size following 21 days of
systemic M.sub.4N treatment. In sum, 100% of the 47 control tumors
increased in size, whereas 53%, or 21 out of 39, M.sub.4N-treated
tumors decreased in size following 21 days of systemic treatment;
of the remaining 18 out of 39 M.sub.4N-treated tumors, although
increasing from their starting tumor size, most exhibited
interrupted growth during the 21 days of treatment. Despite the
significant tumoricidal effect observed for each tumor type, body
weights and the general health of the mice were monitored over the
21 day course of treatment and indicated no toxicity in any of the
mice.
TABLE-US-00003 TABLE 3 Treatment Total # Tumors IOTV DOTV Tumor
Type Control MCF-7 7 7 0 Hep 3B 6 6 0 HT-29 24 24 0 LNCaP 10 10 0
47 (100%) 47 (100%) 0 (0%) Tumor type M.sub.4N MCF-7 10 3 7 Hep 3B
7 5 2 HT-29 11 8 3 LNCaP 11 2 9 39 (100%) 18 (46%) 21 (53%) IOTV:
Increase in Volume from the Original Tumor Volume DOTV: Decrease in
Volume from the Original Tumor Volume
Example 14
Safety Studies in Humans
[0291] In this example, the inventors demonstrated clear safety and
efficacy of NDGA derivative delivery in humans as a therapy for
head and neck cancer. This example describes the results of two
separate clinical studies that spanned range of patient ages,
stages of disease development, and two different treatment methods.
This example demonstrates the following: (1) M.sub.4N can be
delivered by escalating doses up to about 495 mg weekly for three
weeks or at dosages of 20 mg per day for up to five days without
drug-related toxicity. This daily delivery of M.sub.4N can be with
or without concomitant therapy with G.sub.4N at a dose of 20 mg per
day for up to five days and is followed by surgical resection of
the lesion; (2) Both of these treatment methods delivered over 80%
efficacy in terms of induction of necrosis in patients that
completed the treatments; (3) In long-term follow ups of the ex-US
study 64% of patients remained disease free and also free from long
term effects of NDGA-derivative exposure.
[0292] US Phase I Intratumoral Head and Neck Cancer Study:
[0293] A Phase I clinical study has been completed under a US IND.
Mean subject age was 66 years (range 53 to 82 years). Eight male
subjects and one female subject participated. Mean weight was 139
lbs. (range 102 to 219 lbs.). All patients were diagnosed with
refractory head and neck carcinomas.
[0294] Nine (9) subjects were dosed with an intratumoral dose of
M.sub.4N given weekly for three weeks, at doses of 5 mg/cm.sup.3
tumor volume (2 subjects), 10 mg/cm.sup.3 (2 subjects) 15
mg/cm.sup.3 (3 subjects) and 20 mg/cm.sup.3 (2 subjects). Doses up
to 495 mg weekly for three weeks were administered.
[0295] Three subjects completed the study per protocol. Two
subjects died on study from causes considered unlikely to be
related to study medication. One subject withdrew consent after
receiving three doses of M.sub.4N as he could not travel to meet
protocol requirements. One withdrew consent after experienced
severe radiating pain on injection associated with an accidental
perineural dose. One was withdrawn after a single dose as his tumor
was considered to be too close to the carotid artery to allow a
safe second dose. One was withdrawn as a result of tumor
progression. Dosing related adverse events were otherwise minor and
included mild or moderate pain on injection (4 subjects). No other
adverse events were attributed to M.sub.4N administration.
Sporadic, and non-reproducible mild elevations in LFTs were seen in
2 subjects, which resolved while still on therapy. No changes
attributable to drug were seen in hematology parameters.
[0296] Six (6) Serious Adverse Events (SAE) were reported in four
(4) subjects. Serious adverse events included supraventricular
tachycardia (two episodes on separate occasions in one subject),
pneumonia, dehydration and death from tumor progression (one
subject), and death 19 days after study (cause unknown). In all
cases, the SAE's were considered unlikely or not related to study
medication.
[0297] In 5 of 6 subjects receiving three doses, drug related tumor
necrosis occurred after injection. No damage occurred to healthy
tissue surrounding the tumor. Fistula formation developed where
tumors were full thickness. Tumors also were noted to have
softened, or "pancaked", but residual tumor at the margins
continued to grow, suggesting that systemic administration may be
more appropriate. Tumor volume reduction was radiologically
confirmed in three of the six patients completing three doses.
Dosing was generally well tolerated.
Example 15
Safety Studies in Beagle Dogs Following 14-Day Intravenous Infusion
of M.sub.4N
[0298] In this example the Maximal Tolerable Dose (MTD) of two
different formulations of M.sub.4N [Cremaphor-Ethanol (CET) or
Dimethyl Sulfoxide (DMSO)] to male and female beagle dogs was
determined. This example shows that M.sub.4N was safely
administered by intravenous infusion into dogs over four hours at
doses up to 10 mg/kg with a CET vehicle or up to 100 mg/kg with a
DMSO vehicle. Blood levels of up to 14,000 ng/ml M.sub.4N were
achieved with these formulations with minimal toxicity.
[0299] Vascular Access Port (VAP) Implantation Surgery for
M.sub.4N-CET Group
[0300] VAPs were implanted into beagle dogs such that the tip of
the infusion catheter was situated at the level of the superior
vena cava. Dogs were treated prophylactically with an analgesic and
antibiotic on the day of surgery and with antibiotics and/or
analgesics following surgery (according to Gene Logic Inc. SOP Nos.
324.0.2, 325.0.1, and 326.0.2, as appropriate.) Other treatments
were provided as recommended by the staff veterinarian. The
catheter lines were flushed with saline during the postoperative
recovery period with a frequency deemed appropriate by the Study
Director.
[0301] Although VAPs were implanted into dogs that were assigned to
receive infusion of M.sub.4N-DMSO, however, DMSO was found to be
not compatible with the infusion catheter attached to the VAP
inside the animals. Thus the M.sub.4N-DMSO group animals were
administered with M.sub.4N-DMSO with eight intravenous injections
via the non-VAP jugular vein every 30 minutes over a 4-hour period.
This frequency of delivery mimicked the delivery of the test
article using the infusion pump.
TABLE-US-00004 TABLE 4 Group Designation and Dose Levels Number
Dose Infusion Injection of Level Rate Volume Treatment Dogs (mg/kg)
(mL/kg/hr) (mL) Duration M.sub.4N-CET 1M, 1F 0 M (1.7), 2 hours F
(1.3) 1 M (1.7), 2 hours F (1.4) 5 M (0.7), 4 hours F (0.6) 10 M
(1.8), 4 hours F (1.3) M.sub.4N-DMSO.sup.a 1M, 1F 0 M (0.17), 4
hours F (0.12) 10 M (0.16), 4 hours F (0.13) 50 M (0.83), 4 hours F
(0.66) 100 M (1.78), 4 hours F (1.35) M.sub.4N-DMSO.sup.b 1M, 1F
200 M (4.1), ~1 hour F (2.8) .sup.a8 intravenous injections every
30 minutes over 4 hours .sup.bAdditional animals to determine
potential toxicity, M received 3 injections, F received 2
injections
[0302] Animals from the CET group were observed during the entire
infusion period and for at least one hour following end of
infusion. The dogs in the DMSO group were observed throughout the
jugular vein injection period and for at least one hour following
the last (eighth) injection.
[0303] Blood Sample Collection for Toxicokinetic (TK) Analysis
[0304] Blood samples from the CET group animals were collected via
the jugular vein on Study Day (SD)1, SD 3, SD 6, and SD 8 at the
following timepoints: predose, 0.25, 0.5, 1, 2, 4, 8, and 16 hours
following the completion of the approximate 4-hour infusion.
[0305] Blood samples from the DMSO group animals were collected via
the cephalic vein on SD 1, SD 3, SD 6, and SD 8 at the following
timepoints: predose, 0.25, 0.5, 1, 2, 4, 8, and 16 hours following
the final injection dose of M.sub.4N-DMSO.
[0306] Blood samples collected from both groups of animals were
processed for plasma and serum for TK analysis.
[0307] TK Analysis
[0308] The plasma and serum samples were sent to MedTox
Laboratories, the Sponsor's designated laboratory for TK analysis.
TK analysis of M.sub.4N plasma and serum concentration-time data
was performed using a validated method (M200406) by MedTox
Laboratories and analyzed by noncompartmental methods to obtain
estimates of toxicokinetic parameters (where data allow), but not
necessarily limited to, Cmax, Tmax and AUC.
[0309] Study Day 1 (SDI):
[0310] a) M.sub.4N-CET Group
[0311] Male dog: reacted to CET infusion with erythema, hives,
itchiness, emesis, diarrhea, and general lethargy in the first hour
and a half. He began to recover after that, started walking around,
drinking water. He behaved normally soon following end of infusion.
Female dog: reacted similarly to the male dog except without emesis
and diarrhea. Her allergic reactions were also less severe than the
male. She behaved normally soon following end of infusion.
[0312] b) M.sub.4N-DMSO Group
[0313] Male dog: reacted to DMSO with slight erythema, slight
itchiness, otherwise normal. Behavior was normal soon following end
of last injection. Female dog: reacted similarly to the male dog.
Her behavior was normal soon following end of last injection.
[0314] All 4 dogs survived the infusion of their respective vehicle
treatment. They all appeared fine and behaved normally following
treatment.
[0315] Study Day 3 (SD3):
[0316] a) M.sub.4N-CET Group
[0317] Male dog: reacted to M.sub.4N-CET (1 mg/kg) infusion with
slight erythema, hives, itchiness. The reactions this day were
milder than those on SD1. In particular, the animal did not have
emesis, diarrhea, or lethargy, he was more alert than on SD1. He
behaved normally soon following end of infusion. Female dog: her
reactions to the M.sub.4N-CET (1 mg/kg) infusion today was even
milder than those observed on SD1. Her allergic reactions included
mild erythema and itchiness, but she was generally quite alert
throughout the 4-hr infusion period. She behaved normally soon
following end of infusion.
[0318] b) M.sub.4N-DMSO Group
[0319] Male dog: There was no adverse clinical reaction exhibited
by this dog. There was, as expected, some irritation at the
injection sites along the jugular vein.
[0320] Female dog: There was no adverse clinical reaction exhibited
by this dog. There was, as expected, some irritation at the
injection sites along the jugular vein.
[0321] All 4 dogs survived following administration of their
respective test article treatment. They all appeared fine and
behaved normally following treatment.
[0322] Study Day 6 (SD6):
[0323] a) M.sub.4N-CET Group
[0324] Male dog: Similar to the previous two dosing days, this
animal reacted to the infusion with slight erythema, hives, and
itchiness. The intensity of the reactions was certainly no more
than the reactions on SD3. He did not vomit or had diarrhea, was
generally alert throughout the infusion period. He behaved normally
soon following end of infusion.
[0325] Female dog: Consistent with her reactions to previous
dosings, she tolerated today's infusion better than the male dog,
she still had mild erythema and itchiness, but she was quite alert.
She behaved normally soon following end of infusion.
[0326] b) M.sub.4N-DMSO Group
[0327] Male dog: This dog was successfully injected intravenously
with M.sub.4N-DMSO via the non-VAP jugular vein for the first 3
dosing intervals (1/2 hr between doses). As with the previous
dosing days, this dog did not show any adverse clinical signs or
symptoms following each injection. Prior to the fourth injection,
the technicians noticed a swelling "the size of an egg" around the
injection site. Subcutaneous misdose could be ruled out because it
would have been easily detected during the 3rd injection. It was
most likely a hematoma as a result of slow extravasation of blood
through the injection site. This animal did not receive any more
dosing following the third injection, however, blood samples were
collected, the exact time points of the blood collection post-third
injection dose was clearly documented. The hematoma resolved within
two hours and gentle massaging of the injection site area did not
irritate the animal.
[0328] Female dog: This dog was successfully injected with
M.sub.4N-DMSO for the entire 8 repeated injections over 4 hours.
Similar to the previous dosing days, this dog did not show any
adverse clinical signs or symptoms
[0329] Study Day 8 (SD8):
[0330] a) M.sub.4N-CET Group
[0331] Both male and female dogs received full dose. Their
reactions to this high dose were similar to those exhibited in
previous dosing days, which include erythema, hives, and itchiness.
No vomiting or diarrhea was noted. The animals behaved normally
soon following end of infusion.
[0332] b) M.sub.4N-DMSO Group
[0333] Both the male and female dogs received full dose. Their
reactions to the high dose were similar to those on previous dosing
days. There appeared to be more G. I. irritation as both dogs
showed some retching reaction without vomiting, they were more
lethargic than usual. However, both dogs survived the full
high-dose administration regimen and appeared to have recovered
following the end of dosing.
[0334] Additional dose (200 mg/kg) for M.sub.4N-DMSO Group
[0335] Since animals dosed with M.sub.4N-DMSO at 100 mg/Kg did not
show adverse clinical signs or symptoms, two spare dogs (1 male, 1
female) were dosed with M.sub.4N-DMSO at 200 mg/Kg. At 200 mg/Kg,
the female dog experienced difficulty breathing (with nasal
frothing) after only the first of eight doses, she soon collapsed
but was able to recover for the second dose. After the second dose,
her reaction was similar but even more severe. Thus the staff
veterinarian suggested euthanizing the female dog. The male dog was
slightly more tolerant but exhibited similar difficulty breathing
signs and collapsing symptoms. He received a total of three doses
and the staff veterinarian suggested further dosing be stopped.
[0336] There were no post-dose TK analysis for this additional
dosing, all pre-dose blood samples collected today were
discarded.
[0337] TK Analysis
[0338] a) M.sub.4N-CET Group
TABLE-US-00005 TABLE 5 M.sub.4N-CET - SERUM RESULTS (NG/ML) Animal
No./Sex Dose Predose 15 min 30 min 1 hr 2 hr 4 hr 8 hr 16 hr 10828F
1 mg/kg <2 >100 >100 78.59 48.32 39.75 9.88 4.33 10827M 1
mg/kg <2 >100 >100 53.78 32.72 26.65 7.19 4.63 10828F 5
mg/kg <1 >100 >50 >50 >50 34.40 19.45 11.57 10827M 5
mg/kg <1 >50.0 >50 >50 45.35 19.99 12.98 6.71 10828F 10
mg/kg <20.0 >1000 >1000 705.47 285.10 187.82 133.50 52.94
10827M 10 mg/kg 63.76 >1000 >1000 783.86 431.21 158.20 117.00
60.36
In general, intravenous infusion of M.sub.4N-CET at different dose
levels for 4 hours resulted in extremely high serum concentrations
at the early time points and peaked at 30 minutes following end of
infusion (Table 5). The serum concentrations of the test article
reduced over the next 15 hours.
[0339] b) M.sub.4N-DMSO Group
TABLE-US-00006 TABLE 6 M.sub.4N-DMSO - SERUM RESULTS (NG/ML) Animal
No./Sex Dose Predose 15 min 30 min 1 hr 2 hr 4 hr 8 hr 16 hr 10831F
10 mg/kg <2 594.98 398.95 436.92 238.20 97.92 43.80 38.39 10832M
10 mg/kg <2 516.8 533.07 348.7 252.86 317.13 78.87 56.27 10831F
50 mg/kg 3.49 1136.51 474.95 673.4 241 144 101 58.8 10832M 50 mg/kg
19.91 NR NR NR 234.15 79.11 65.79 45.8 10831F 100 mg/kg 21.47
8688.10 8163.68 7696.48 2624.2 1021.05 459.82 222.22 10832M 100
mg/kg 38.22 10477 14088 7498.88 3878.86 3468.19 814.24 593.83
[0340] In general, repeated intravenous injection of M.sub.4N-DMSO
at different dose levels for 4 hours resulted in extremely high
serum concentrations. The serum concentration data reported in
Table 6 are the results following systematic dilution of the serum
to accommodate detection range. The results showed that in general,
the serum concentration of M.sub.4N-DMSO was high in the early time
points and peaked at 30 minutes following the last injection. The
serum concentrations of the test article reduced over the next 15
hours. Based on the serum concentrations from this group, the
half-life of M.sub.4N-DMSO, when administered by repeated
intravenous injection, was approximately 1.5 to 2 hours. It is
noteworthy that from the pre-dose serum concentrations of
M.sub.4N-DMSO over the course of this MTD phase, there was a slight
build-up of the test article in the blood, but this retention was
generally less than 0.3% of the highest serum concentration.
[0341] The purpose of the MTD phase of this study was to determine
the maximum tolerable dose of two different formulations of
M.sub.4N (Cremaphor-Ethanol or DMSO) to male and female beagle
dogs. The group of animals that received M.sub.4N-CET reacted with
itchiness, erythema, hives, and sleepiness; clinical signs and
symptoms consistent with the effects of Cremaphor-Ethanol. Animals
that received repeated injections of M.sub.4N-DMSO showed some
irritation at the injection site and minor retching at 100 mg/kg.
However, both animals collapsed following 2 or 3 injections of
M.sub.4N-DMSO at 200 mg/kg. TK analysis from this group suggested a
half life for M.sub.4N-DMSO in the range of 1.5-2 hours. There was
minor build up of the test article over the course of the MTD
phase, however, this retention only amounted to less than 0.3% of
the maximum serum concentration. In conclusion, the MTD phase of
this study was a success as the dose level that resulted in
significant adverse clinical signs and symptoms was identified,
thus the objective of this phase was achieved.
[0342] Treatment of Therapy-Refractory Acute Lymphoblastic Leukemia
with NDGA Derivatives.
[0343] NDGA derivatives can be used as a treatment for acute
lymphoblastic leukemia (ALL). Such use can be assessed via a method
similar to that described by Uckun, F. M. et al. (1999). For
example, patients with ALL having relapses following frontline or
salvage chemotherapy or having failed induction chemotherapy can be
subject of treatment with the NDGA derivative.
[0344] In one example, the NDGA derivative, G4N, is formulated as a
sterile solution, for example, 1 mg/ml in 150 mM sodium chloride
and 40 mM sodium phosphate (pH 7.4). For intravenous
administration, the drug is further diluted in 10 ml/kg (up to
about 100 ml) normal saline. Each treatment consists of a
continuous infusion for a period of time depending on the half-life
of the particular NDGA derivative, for example, a 1-hr infusion,
given 15-20 minutes after premedication with standard doses of
diphenhydramine (Benadryl) and acetaminophen (Tylenol). Patients
are treated with one or two courses of therapy, each comprising
either 10 consecutive days of treatment or three weekly cycles of 3
consecutive days each for a total of nine doses. The doses to be
given depend on the characteristics of the patients and the status
of the diseases and can be, for example, one of 0.1 mg/kg/day, or
0.18 mg/kg/day, or 0.32 mg/kg/day or higher.
[0345] The successfulness of NDGA therapy for the treatment of ALL
is summarized by classifying patient cases as complete remission,
partial response, progressive disease, or stable disease. Complete
remission ("CR") of ALL in response to NDGA derivative treatment is
defined as the achievement of M1 bone marrow status (<5% blasts)
by day 28 of therapy with a granulocyte count of
1.times.10.sup.3/.mu.l, a platelet count of
100.times.10.sup.3/.mu.l (>50,000/.mu.l without transfusions for
patients post-BMT), a hemoglobin level of 10 g/dl, and absence of
circulating leukemia cells in the peripheral blood or any evidence
of extramedullary disease. Partial response ("PR") is defined as a
complete disappearance of peripheral blasts and achievement of M2
bone marrow status (5-25% blasts) by day 28 of therapy. Progressive
disease ("PD") is defined as an increase of at least 25% in the
absolute number of circulating blasts, or development of
extramedullary disease. Stable disease ("SD") is defined as a lack
of change in status of any of the parameters that would result in
CR, PR, or PD. NDGA derivatives are seen to have some inhibitory
effect on the development of PD.
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[0394] It will be appreciated by those skilled in the art that
changes could be made to the embodiments described above without
departing from the broad inventive concept thereof. It is
understood, therefore, that this invention is not limited to the
particular embodiments disclosed, but it is intended to cover
modifications within the spirit and scope of the present invention
as defined by the appended claims.
* * * * *