U.S. patent application number 14/282336 was filed with the patent office on 2014-09-11 for co-enzyme q10 formulations and methods of use.
This patent application is currently assigned to UNIVERSITY OF MIAMI. The applicant listed for this patent is UNIVERSITY OF MIAMI. Invention is credited to Sung Lan Hsia, Jie Li, Niven Rajin Narain, Indushekhar Persaud, Kathryn J. Russell, Karrune V. Woan.
Application Number | 20140255372 14/282336 |
Document ID | / |
Family ID | 34807174 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140255372 |
Kind Code |
A1 |
Hsia; Sung Lan ; et
al. |
September 11, 2014 |
CO-ENZYME Q10 FORMULATIONS AND METHODS OF USE
Abstract
Topical formulations of CoQ10 reduce the rate of tumor growth in
an animal subject. In the experiments described herein, CoQ10 was
shown to increase the rate of apoptosis in a culture of skin cancer
cells but not normal cells. Moreover, treatment of tumor-bearing
animals with a topical formulation of CoQ10 was shown to
dramatically reduce the rate of tumor growth in the animals.
Inventors: |
Hsia; Sung Lan; (Miami,
FL) ; Narain; Niven Rajin; (Cambridge, MA) ;
Li; Jie; (Miami, FL) ; Russell; Kathryn J.;
(New York, NY) ; Woan; Karrune V.; (Gainesville,
FL) ; Persaud; Indushekhar; (Homestead, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF MIAMI |
Miami |
FL |
US |
|
|
Assignee: |
UNIVERSITY OF MIAMI
Miami
FL
|
Family ID: |
34807174 |
Appl. No.: |
14/282336 |
Filed: |
May 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14031706 |
Sep 19, 2013 |
8771680 |
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14282336 |
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13791313 |
Mar 8, 2013 |
8586030 |
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14031706 |
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13366224 |
Feb 3, 2012 |
8562976 |
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13791313 |
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10597378 |
Aug 21, 2008 |
8147825 |
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PCT/US2005/001581 |
Jan 21, 2005 |
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13366224 |
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60538319 |
Jan 22, 2004 |
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Current U.S.
Class: |
424/94.1 ;
435/375; 514/690 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/02 20180101; A61K 47/24 20130101; A61P 35/04 20180101; A61K
9/06 20130101; A61K 36/53 20130101; A61P 35/00 20180101; A61K 45/06
20130101; A61K 31/122 20130101; A61P 9/10 20180101; A61P 9/00
20180101; A61K 9/0014 20130101; A61K 31/351 20130101; A61P 17/00
20180101; A61P 29/00 20180101; A61K 31/122 20130101; A61K 2300/00
20130101; A61K 36/53 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/94.1 ;
514/690; 435/375 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 45/06 20060101 A61K045/06; A61K 31/122 20060101
A61K031/122 |
Claims
1-7. (canceled)
8. A method of treating a cancer patient, comprising: administering
to a patient in need thereof, a composition comprising a
therapeutically effective amount of Coenzyme Q10; contacting a
tumor cell with the composition resulting in the lysis of the tumor
cell; thereby treating the cancer patient.
9. The method of claim 8, wherein the composition comprises about
1% up to 25% w/w of Coenzyme Q10.
10. The method of claim 8, wherein the composition comprises about
1% to about 20% w/w of Coenzyme Q10.
11. The method of claim 8, wherein the composition comprising the
Coenzyme Q10 is formulated as a topical cream.
12. The method of claim 8, wherein a therapeutic effective amount
of the Coenzyme Q10 composition is administered with one or more
chemotherapeutic agents.
13. The method of claim 12, wherein the chemotherapeutic agent can
be co-administered, precede, or administered after the composition
comprising a therapeutic effective amount of Coenzyme Q10.
14. The method of claim 12, wherein the chemotherapeutic agent is
selected from the group consisting of cyclophosphamide (CTX, 25
mg/kg/day, p.o.), taxanes (paclitaxel or docetaxel), busulfan,
cisplatin, cyclophosphamide, methotrexate, daunorubicin,
doxorubicin, melphalan, cladribine, vincristine, vinblastine, and
chlorambucil.
15. The method of claim 8, wherein treatment results in inhibition
of tumor cell growth.
16. A method for inhibiting tumor cell growth in a subject, the
method comprising administering to the subject a pharmaceutical
composition comprising CoQ10.
17. The method of claim 16, wherein the pharmaceutical composition
comprises between about 1% and 25% w/w of coenzyme Q10.
18. The method of claim 16, wherein the pharmaceutical composition
comprises about 1% up to 25% w/w of Coenzyme Q10.
19. The method of claim 16, wherein the pharmaceutical composition
comprises about 1% to about 20% w/w of Coenzyme Q10.
20. A method of inducing apoptosis in a tumor cell, the method
comprising administering a pharmaceutical composition comprising
coenzyme Q10.
21. The method of claim 20, wherein the pharmaceutical composition
comprises about 1% up to 25% w/w of Coenzyme Q10.
22. The method of claim 20, wherein the pharmaceutical composition
comprises about 1% to about 20% w/w of Coenzyme Q10.
23. The method of claim 20, wherein the pharmaceutical composition
induces apoptosis in at least about 30% of tumor cells as measured
by mitochondrial membrane dye assay and/or Annexin-VPE assay.
24. The method of claim 20, wherein the pharmaceutical composition
induces apoptosis in about 50% of tumor cells as measured by
mitochondrial membrane dye assay and/or Annexin-VPE assay.
25. The method of claim 20, wherein the pharmaceutical composition
induces apoptosis in about 60% of tumor cells as measured by
mitochondrial membrane dye assay and/or Annexin-VPE assay.
26. The method of claim 20, wherein the pharmaceutical composition
induces apoptosis in about 75% of tumor cells as measured by
mitochondrial membrane dye assay and/or Annexin-VPE assay.
27. The method of claim 20, wherein the pharmaceutical composition
induces apoptosis in about 90% of tumor cells as measured by
mitochondrial membrane dye assay and/or Annexin-VPE assay.
28. The method of claim 20, wherein the pharmaceutical composition
induces apoptosis in about 99.9% of tumor cells as measured by
mitochondrial membrane dye assay and/or Annexin-VPE assay.
29. A method of inhibiting angiogenesis in a tumor, the method
comprising contacting a tumor with a pharmaceutical composition
comprising coenzyme Q10.
30. The method of claim 29, wherein the pharmaceutical composition
comprises about 1% up to 25% w/w of Coenzyme Q10.
31. The method of claim 29, wherein the pharmaceutical composition
comprises about 1% to about 20% w/w of Coenzyme Q10.
33-37. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention provides pharmaceutical compositions
comprising co-enzyme Q10 (CoQ10) and methods of using CoQ10 for
treatment of cancer, selective reduction of cancer cell growth,
induction of apoptosis in cancer cells and inhibition of tumor
mediated angiogenesis.
BACKGROUND
[0002] Cancer is presently one of the leading causes of death in
developed nations. Although recent research has vastly increased
our understanding of many of the molecular mechanisms of
tumorigenesis and has provided numerous new avenues for the
treatment of cancer, standard treatments for most malignancies
remain gross resection, chemotherapy, and radiotherapy. While
increasingly successful, each of these treatments still causes
numerous undesired side effects. For example, surgery results in
pain, traumatic injury to healthy tissue, and scarring.
Radiotherapy and chemotherapy cause nausea, immune suppression,
gastric ulceration and secondary tumorigenesis.
SUMMARY
[0003] The invention relates to the discovery that topical
formulations of CoQ10 can reduce the rate of tumor growth in an
animal subject. In the experiments described herein, CoQ10 was
shown to increase the rate of apoptosis in a culture of skin cancer
cells but not normal cells. Moreover, treatment of tumor-bearing
animals with a topical formulation of CoQ10 was shown to
dramatically reduce the rate of tumor growth in the animals.
[0004] CoQ10 formulated for oral delivery has previously been used
as a dietary supplement. Orally administered CoQ10 has, however,
been shown to accumulate in the liver-diminishing its systemic
availability. The anti-tumor responses observed with topically
applied CoQ10 may relate to its higher bioavailability compared to
dietary supplement forms of the CoQ10.
[0005] Accordingly, the invention features a method for reducing
the rate of tumor cell growth or increasing the rate of apoptosis
in tumor cells in a subject. The method includes the steps of
providing a subject having a plurality of tumor cells and
administering to the subject a composition comprising an effective
amount of CoQ10 and a pharmaceutically acceptable carrier.
[0006] In another aspect, the invention features a composition
comprising an effective amount of CoQ10 and a pharmaceutically
acceptable carrier.
[0007] In a preferred embodiment, the composition is a topical
formulation of CoQ10 that includes at least about 0.01% by weight
CoQ 10 up to 30% by weight (w/w) of CoQ10 and a carrier suitable
for delivering the CoQ10 topically. Preferably, the pharmaceutical
composition comprises as an active ingredient CoQ10 and a
pharmaceutically acceptable carrier. The composition comprising,
Coenzyme Q10, phospholipon 90, glycerol, butylated hydroxytoluene
(BHT), ethanol, medium chain triglycerides (MCT) and lavender.
Preferably, the phospholipon 90 is phospholipon 90G and/or
phospholipon 90H.
[0008] In a preferred embodiment, the pharmaceutical composition
comprises at least about 0.01% to about 30% (w/w) of Coenzyme Q10.
Preferably, the pharmaceutical composition between about 1% to
about 25% (w/w) of Coenzyme Q10.
[0009] In another preferred embodiment, the invention provides a
method of treating a cancer patient, comprising:
[0010] administering to a patient in need thereof, a composition
comprising a therapeutically
[0011] effective amount of Coenzyme Q10;
[0012] contacting a tumor cell with the composition resulting in
the lysis of the tumor cell;
[0013] thereby treating the cancer patient.
Preferably, the pharmaceutical composition comprises at least about
0.01% up to 30% w/w of Coenzyme Q10, preferably, the pharmaceutical
composition comprises about 1% to about 25% w/w of Coenzyme
Q10.
[0014] In another preferred embodiment, the pharmaceutical
composition is formulated in a topical cream with optional
transdermal enhancers.
[0015] In other preferred embodiments, a therapeutically effective
amount of the Coenzyme Q10 composition is administered with one or
more chemotherapeutic agents. These chemotherapeutic agents can be
co-administered, precede, or administered after the Coenzyme Q10.
Non-limiting examples of chemotherapeutic agents include, but not
limited to: cyclophosphamide (CTX, 25 mg/kg/day, p.o.), taxanes
(paclitaxel or docetaxel), busulfan, cisplatin, cyclophosphamide,
methotrexate, daunorubicin, doxorubicin, melphalan, cladribine,
vincristine, vinblastine, and chlorambucil.
[0016] In another preferred embodiment, the pharmaceutical
composition, Coenzyme Q10 composition inhibits the tumor cell
growth in a subject, and the method comprises administering to the
subject a pharmaceutical composition comprising a therapeutically
effective amount of CoQ10. Preferably, the therapeutically
effective amount of Coenzyme Q10 in the pharmaceutical composition
comprises between about 0.01% and 30% w/w of coenzyme Q10.
Inhibition of tumor cell growth refers to one or more of the
following effects: (1) inhibition, to some extent, of tumor growth,
including, (i) slowing down and (ii) complete growth arrest; (2)
reduction in the number of tumor cells; (3) maintaining tumor size;
(4) reduction in tumor size; (5) inhibition, including (i)
reduction, (ii) slowing down or (iii) complete prevention, of tumor
cell infiltration into peripheral organs; (6) inhibition, including
(i) reduction, (ii) slowing down or (iii) complete prevention, of
metastasis; (7) enhancement of anti-tumor immune response, which
may result in (i) maintaining tumor size, (ii) reducing tumor size,
(iii) slowing the growth of a tumor, (iv) reducing, slowing or
preventing invasion and/or (8) relief, to some extent, of the
severity or number of one or more symptoms associated with the
disorder.
[0017] In another preferred embodiment, the invention provides a
method of inducing apoptosis selectively in a tumor cell, the
method comprising administering a pharmaceutical composition
comprising coenzyme Q10 as measured in standard assays. Preferably,
the pharmaceutical composition comprises at least about 0.01% up to
30% w/w of Coenzyme Q10. Methods for measuring apoptosis include
but not limited to mitochondrial membrane dye assays and/or
Annexin-VPE assays. In a preferred embodiment, the pharmaceutical
composition induces apoptosis in at least about 30% of tumor cells
as measured by mitochondrial membrane dye assay and/or Annexin-VPE
assay. Preferably, the pharmaceutical composition induces apoptosis
in about 60% of tumor cells as measured by mitochondrial membrane
dye assay and/or Annexin-VPE assay, more preferably, the
pharmaceutical composition induces apoptosis in about 75% of tumor
cells as measured by mitochondrial membrane dye assay and/or
Annexin-VPE assay, more preferably, the pharmaceutical composition
induces apoptosis in about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% and 100% of tumor cells as measured by mitochondrial
membrane dye assay and/or Annexin-VPE assay.
[0018] In another preferred embodiment, the invention provides a
method of inhibiting angiogenesis in a tumor, the method comprising
contacting a tumor with a pharmaceutical composition comprising
coenzyme Q10. Preferably, the pharmaceutical composition comprises
at least about 0.01% up to 30% w/w of Coenzyme Q10.
[0019] Additional uses for the present compounds include use in the
treatment of atherosclerosis, inflammation, and as an
anti-angiogenic agent, especially to treat cancers, particularly
solid cancers such as cancers residing in the lung, breast, liver,
brain or other tissue.
[0020] Unless otherwise defined, all technical terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art to which this invention belongs. Commonly
understood definitions of medical terms can be found in Thomas
Lathrop Stedman, Stedman's Medical Dictionary, Lippincott, Williams
& Wilkins: Philadelphia, Pa., 2000.
[0021] All publications, patent applications, patents and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions will control. The particular embodiments
discussed below are illustrative only and not intended to be
limiting.
[0022] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The invention is pointed out with particularity in the
appended claims. The above and further advantages of this invention
may be better understood by referring to the following description
taken in conjunction with the accompanying drawings, in which:
[0024] FIG. 1. is a series of photomicrographs showing the effect
of CoQ10 on human melanoma cells (SKMEL28) in an in vitro
culture.
[0025] FIG. 2 is a graph showing that CoQ10 reduces the
proliferation of a human melanoma cell line (SKMEL28) in a 36 hour
in vitro culture.
[0026] FIG. 3 is a graph showing that CoQ10 reduces the
proliferation of a human melanoma cell line (SKMEL28) in a 48 hour
in vitro culture.
[0027] FIG. 4 is a graph showing that the vehicle control does not
reduce the proliferation of a human melanoma cell line (SKMEL28) in
a 48 hour in vitro culture.
[0028] FIG. 5 is a graph comparing the effect of CoQ10 on apoptosis
between human melanoma and neonatal fibroblasts in an in vitro
culture.
[0029] FIG. 6 is a graph showing that CoQ10 reduces the
proliferation of squamous carcinoma cells in a 48 hour in vitro
culture.
[0030] FIG. 7 is a graph showing that CoQ10 reduces the
proliferation of human neonatal fibroblasts in a 48 hour in vitro
culture.
[0031] FIG. 8 is a graph showing that CoQ10 increases the
proliferation of human neonatal keratinocytes in a 48 hour in vitro
culture.
[0032] FIG. 9 is a graph showing that CoQ10 reduces the
proliferation of a breast adenocarcinoma cell line (MCF-7) in a 48
hour in vitro culture. The MCF-7 cell line expresses the WNT7B
oncogene and contains the Tx-4 oncogene.
[0033] FIG. 10 is a graph showing that CoQ10 reduces the
proliferation of a breast adenocarcinoma cell line (MCF-7) in a 72
hour in vitro culture.
[0034] FIG. 11 is a photograph showing induced tumors in control
and CoQ10-treated mice after treatment with topical formulation of
CoQ10 for 30 days.
[0035] FIG. 12 is a photograph showing induced tumors in control
and CoQ10-treated mice after treatment with topical formulation of
CoQ10 for 30 days.
[0036] FIG. 13 is a photograph showing tumors excised from control
and CoQ10-treated mice.
[0037] FIG. 14 is a graph showing the effect of CoQ10
administration on tumor size on mice treated with CoQ10 or control
for 30 days. Average tumor mass for the control vs. treatment group
decreased by 52.3% and 54.0%, respectively.
[0038] FIG. 15 is a series of photomicrographs showing the effect
of CoQ10 on human breast adenocarcinoma cells (SK-BR-3) in an in
vitro culture. The SK-BR-3 cell line overexpresses the Her2/c-erb-2
genes, gene product (ATCC).
[0039] FIG. 16 is a graph showing that CoQ10 reduces the
proliferation of a human breast adenocarcinoma cell line (SK-BR-3)
in a 48 hour in vitro culture. The SK-BR-3 cell line overexpresses
the Her2/c-erb-2 genes gene product (ATCC).
[0040] FIG. 17 is a graph showing that CoQ10 reduces the
proliferation of a human breast adenocarcinoma cell line (SK-BR-3)
in a 72 hour in vitro culture. The SK-BR-3 cell line overexpresses
the Her2/c-erb-2 genes gene product (ATCC).
[0041] FIG. 18 is a graph showing that CoQ10 reduces the
proliferation of a human breast adenocarcinoma cell line
(MDA-MB-468) in a 48 hour in vitro culture. The MDA-MB-468 cell
line has a mutation in the p53 gene (ATCC).
[0042] FIG. 19 is a graph showing that CoQ10 reduces the
proliferation of a human breast adenocarcinoma cell line
(MDA-MB-468) in a 72 hour in vitro culture. The MDA-MB-468 cell
line has a mutation in the p53 gene (ATCC).
[0043] FIG. 20 is a graph showing that CoQ10 reduces the
proliferation of a human breast adenocarcinoma cell line (BT-20) in
a 48 hour in vitro culture. The BT-20 cell line expresses the WNT7B
and WNT3 oncogene (ATCC).
[0044] FIG. 21 is a graph showing that CoQ10 reduces the
proliferation of a human breast adenocarcinoma cell line (BT-20) in
a 72 hour in vitro culture. The BT-20 cell line expresses the WNT7B
and WNT3 oncogene (ATCC).
[0045] FIG. 22 is a graph showing that CoQ10 reduces the
proliferation of a human hepatocellular carcinoma cell line (Hep
3B) in a 48 hour in vitro culture.
[0046] FIG. 23 is a graph showing that CoQ10 reduces the
proliferation of a human hepatocellular carcinoma cell line (Hep
3B) in a 72 hour in vitro culture.
[0047] FIG. 24 is a graph showing that CoQ10 reduces the
proliferation of a human osteosarcoma cell line (143B) in a 48 hour
in vitro culture.
[0048] FIG. 25 is a graph showing that CoQ10 reduces the
proliferation of a human osteosarcoma cell line (143B) in a 72 hour
in vitro culture.
[0049] FIG. 26 is a graph showing that CoQ10 reduces the
proliferation of a human prostatic adenocarcinoma cell line (PC-3)
in a 48 hour in vitro culture.
[0050] FIG. 27 is a graph showing that CoQ10 reduces the
proliferation of a human prostatic adenocarcinoma cell line (PC-3)
in a 72 hour in vitro culture.
[0051] FIG. 28 is a graph showing the effect of CoQ10 on
mitochondrial polarization (an indicator of apoptosis) of a human
prostatic adenocarcinoma cell line (PC-3) in a 24 hour in vitro
culture. PC-3 cell cultures were treated with Q10 at 0.05, 0.1 and
0.2 mM concentrations for 24 h and then treated with JC-1, at 10
microgram/ml., for 30 min. Uptake and levels of green fluorescence
was measured in a flow cytometer, FL1 (green fluor.). Note: A
significant increase in the green fluorescence was observed in 0.2
mM Q10 treated cells (yellow graph).
[0052] FIGS. 29A and 29B are photographs showing inhibition of
tumor-mediated angiogenesis in tissues by a composition comprising
CoQ10 (FIG. 29B) as compared to a control in the absence of a
composition comprising CoQ10.
DETAILED DESCRIPTION
[0053] The invention provides compositions and methods for reducing
the rate of tumor cell growth or increasing the rate of tumor cell
apoptosis. Compositions of the invention include as an anti-tumor
agent a therapeutically effective amount of CoQ10 and a carrier. A
preferred composition of the invention is a topical formulation of
CoQ10 comprising at least about 1% CoQ10 and a carrier that
facilitates topical delivery of CoQ10. A most preferred composition
of the invention is a topical formulation of CoQ10 comprising
between about 1% and 15% CoQ10 and a carrier that facilitates
topical delivery of CoQ10. Methods of the invention for killing a
tumor cell or reducing its growth rate include the step of
contacting the cell with an effective concentration of CoQ10.
[0054] The below described preferred embodiments illustrate
adaptations of these compositions and methods. Nonetheless, from
the description of these embodiments, other aspects of the
invention can be made and/or practiced based on the description
provided below.
[0055] Before the present invention is disclosed and described, it
is to be understood that this invention is not limited to the
particular structures, process steps, or materials disclosed
herein, but is extended to equivalents thereof as would be
recognized by those ordinarily skilled in the relevant arts. It
should also be understood that terminology employed herein is used
for the purpose of describing particular embodiments only and is
not intended to be limiting.
DEFINITIONS
[0056] In accordance with the present invention and as used herein,
the following terms are defined with the following meanings, unless
explicitly stated otherwise.
[0057] As used herein, "a", "an," and "the" include plural
references unless the context clearly dictates otherwise.
[0058] As used herein, a "pharmaceutically acceptable" component is
one that is suitable for use with humans and/or animals without
undue adverse side effects (such as toxicity, irritation, and
allergic response) commensurate with a reasonable benefit/risk
ratio.
[0059] As used herein, the term "safe and therapeutic effective
amount" refers to the quantity of a component which is sufficient
to yield a desired therapeutic response without undue adverse side
effects (such as toxicity, irritation, or allergic response)
commensurate with a reasonable benefit/risk ratio when used in the
manner of this invention. By "therapeutically effective amount" is
meant an amount of a compound of the present invention effective to
yield the desired therapeutic response. For example, an amount
effective to delay the growth of or to cause a cancer, either a
sarcoma or lymphoma, or to shrink the cancer or prevent metastasis.
The specific safe and effective amount or therapeutically effective
amount will vary with such factors as the particular condition
being treated, the physical condition of the patient, the type of
mammal or animal being treated, the duration of the treatment, the
nature of concurrent therapy (if any), and the specific
formulations employed and the structure of the compounds or its
derivatives.
[0060] As used herein, a "pharmaceutical salt" include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines; alkali or organic salts of acidic residues such as
carboxylic acids. Preferably the salts are made using an organic or
inorganic acid. These preferred acid salts are chlorides, bromides,
sulfates, nitrates, phosphates, sulfonates, formates, tartrates,
maleates, malates, citrates, benzoates, salicylates, ascorbates,
and the like. The most preferred salt is the hydrochloride
salt.
[0061] As used herein, "cancer" refers to all types of cancer or
neoplasm or malignant tumors found in mammals, including, but not
limited to: leukemias, lymphomas, melanomas, carcinomas and
sarcomas. In preferred embodiments, the CoQ10 compositions are used
for treatment, of various types of breast cancer; prostate cancer;
liver cancer; bone cancer. However, treatment using the CoQ10
compositions are not limited to these types of cancers.
[0062] Examples of cancers are cancer of the brain, breast,
pancreas, cervix, colon, head and neck, kidney, lung, non-small
cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus
and Medulloblastoma. As used herein, the terms "cancer,"
"neoplasm," and "tumor," are used interchangeably and in either the
singular or plural form, refer to cells that have undergone a
malignant transformation that makes them pathological to the host
organism. Primary cancer cells (that is, cells obtained from near
the site of malignant transformation) can be readily distinguished
from non-cancerous cells by well-established techniques,
particularly histological examination. The definition of a cancer
cell, as used herein, includes not only a primary cancer cell, but
any cell derived from a cancer cell ancestor. This includes
metastasized cancer cells, and in vitro cultures and cell lines
derived from cancer cells. When referring to a type of cancer that
normally manifests as a solid tumor, a "clinically detectable"
tumor is one that is detectable on the basis of tumor mass; e.g.,
by procedures such as CAT scan, MR imaging, X-ray, ultrasound or
palpation, and/or which is detectable because of the expression of
one or more cancer-specific antigens in a sample obtainable from a
patient.
[0063] The term "sarcoma" generally refers to a tumor which is made
up of a substance like the embryonic connective tissue and is
generally composed of closely packed cells embedded in a fibrillar
or homogeneous substance. Examples of sarcomas which can be treated
with the present compositions and optionally a potentiator and/or
chemotherapeutic agent include, but not limited to a
chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma,
myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma,
liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,
botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal
sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal
sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,
giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,
idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic
sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells,
Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma,
angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma,
parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic
sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
[0064] The term "melanoma" is taken to mean a tumor arising from
the melanocytic system of the skin and other organs. Melanomas
which can be treated with the compositions of the invention and
optionally a potentiator and/or another chemotherapeutic agent
include but not limited to, for example, acral-lentiginous
melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's
melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma,
lentigo maligna melanoma, malignant melanoma, nodular melanoma,
subungal melanoma, and superficial spreading melanoma.
[0065] The term "carcinoma" refers to a malignant new growth made
up of epithelial cells tending to infiltrate the surrounding
tissues and give rise to metastases. Carcinomas which can be
treated with the compositions of the invention and optionally a
potentiator and/or a chemotherapeutic agent include but not limited
to, for example, acinar carcinoma, acinous carcinoma, adenocystic
carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,
carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell
carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid
carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,
bronchiolar carcinoma, bronchogenic carcinoma, cerebriform
carcinoma, cholangiocellular carcinoma, chorionic carcinoma,
colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform
carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical
carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma
durum, embryonal carcinoma, encephaloid carcinoma, epiermoid
carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,
carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,
gelatinous carcinoma, giant cell carcinoma, carcinoma
gigantocellulare, glandular carcinoma, granulosa cell carcinoma,
hair-matrix carcinoma, hematoid carcinoma, hepatocellular
carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid
carcinoma, infantile embryonal carcinoma, carcinoma in situ,
intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's
carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma,
lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma,
lymphoepithelial carcinoma, carcinoma medullare, medullary
carcinoma, melanotic carcinoma, carcinoma molle, mucinous
carcinoma, carcinoma muciparum, carcinoma mucocellulare,
mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma,
carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell
carcinoma, carcinoma ossificans, osteoid carcinoma, papillary
carcinoma, periportal carcinoma, preinvasive carcinoma, prickle
cell carcinoma, pultaceous carcinoma, renal cell carcinoma of
kidney, reserve cell carcinoma, carcinoma sarcomatodes,
schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti,
signet-ring cell carcinoma, carcinoma simplex, small-cell
carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle
cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous
cell carcinoma, string carcinoma, carcinoma telangiectaticum,
carcinoma telangiectodes, transitional cell carcinoma, carcinoma
tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma
villosum.
[0066] Additional cancers which can be treated with the
compositions of the invention include, for example, Hodgkin's
Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,
breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma,
primary thrombocytosis, primary macroglobulinemia, small-cell lung
tumors, primary brain tumors, stomach cancer, colon cancer,
malignant pancreatic insulanoma, malignant carcinoid, urinary
bladder cancer, premalignant skin lesions, testicular cancer,
lymphomas, thyroid cancer, neuroblastoma, esophageal cancer,
genitourinary tract cancer, malignant hypercalcemia, cervical
cancer, endometrial cancer, adrenal cortical cancer, and prostate
cancer.
[0067] "Diagnostic" or "diagnosed" means identifying the presence
or nature of a pathologic condition. Diagnostic methods differ in
their sensitivity and specificity. The "sensitivity" of a
diagnostic assay is the percentage of diseased individuals who test
positive (percent of "true positives"). Diseased individuals not
detected by the assay are "false negatives." Subjects who are not
diseased and who test negative in the assay, are termed "true
negatives." The "specificity" of a diagnostic assay is 1 minus the
false positive rate, where the "false positive" rate is defined as
the proportion of those without the disease who test positive.
While a particular diagnostic method may not provide a definitive
diagnosis of a condition, it suffices if the method provides a
positive indication that aids in diagnosis.
[0068] The terms "patient" or "individual" are used interchangeably
herein, and refers to a mammalian subject to be treated, with human
patients being preferred. In some cases, the methods of the
invention find use in experimental animals, in veterinary
application, and in the development of animal models for disease,
including, but not limited to, rodents including mice, rats, and
hamsters; and primates.
[0069] "Sample" is used herein in its broadest sense. A sample
comprising polynucleotides, polypeptides, peptides, antibodies and
the like may comprise a bodily fluid; a soluble fraction of a cell
preparation, or media in which cells were grown; a chromosome, an
organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound
to a substrate; a cell; a tissue; a tissue print; a fingerprint,
skin or hair; and the like.
[0070] "Treatment" is an intervention performed with the intention
of preventing the development or altering the pathology or symptoms
of a disorder. Accordingly, "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures. Those in need
of treatment include those already with the disorder as well as
those in which the disorder is to be prevented. In tumor (e.g.,
cancer) treatment, a therapeutic agent may directly decrease the
pathology of tumor cells, or render the tumor cells more
susceptible to treatment by other therapeutic agents, e.g.,
radiation and/or chemotherapy. As used herein, "ameliorated" or
"treatment" refers to a symptom which is approaches a normalized
value (for example a value obtained in a healthy patient or
individual), e.g., is less than 50% different from a normalized
value, preferably is less than about 25% different from a
normalized value, more preferably, is less than 10% different from
a normalized value, and still more preferably, is not significantly
different from a normalized value as determined using routine
statistical tests. For example, the "treatment of cancer" or "tumor
cells", refers to one or more of the following effects: (1)
inhibition, to some extent, of tumor growth, including, (i) slowing
down and (ii) complete growth arrest; (2) reduction in the number
of tumor cells; (3) maintaining tumor size; (4) reduction in tumor
size; (5) inhibition, including (i) reduction, (ii) slowing down or
(iii) complete prevention, of tumor cell infiltration into
peripheral organs; (6) inhibition, including (i) reduction, (ii)
slowing down or (iii) complete prevention, of metastasis; (7)
enhancement of anti-tumor immune response, which may result in (i)
maintaining tumor size, (ii) reducing tumor size, (iii) slowing the
growth of a tumor, (iv) reducing, slowing or preventing invasion
and/or (8) relief, to some extent, of the severity or number of one
or more symptoms associated with the disorder.
[0071] As used herein, "an ameliorated symptom" or "treated
symptom" refers to a symptom which is approaches a normalized
value, e.g., is less than 50% different from a normalized value,
preferably is less than about 25% different from a normalized
value, more preferably, is less than 10% different from a
normalized value, and still more preferably, is not significantly
different from a normalized value as determined using routine
statistical tests.
[0072] A "chemokine" is a small cytokine involved in the migration
and activation of cells, including phagocytes and lymphocytes, and
plays a role in inflammatory responses.
[0073] A "cytokine" is a protein made by a cell that affect the
behavior of other cells through a "cytokine receptor" on the
surface of the cells the cytokine effects. Cytokines manufactured
by lymphocytes are sometimes termed "lymphokines." Cytokines are
also characterized as Type I (e.g. IL-2 and IFN-.gamma.) and Type
II (e.g. IL-4 and IL-10).
[0074] By the term "modulate," it is meant that any of the
mentioned activities, are, e.g., increased, enhanced, increased,
agonized (acts as an agonist), promoted, decreased, reduced,
suppressed blocked, or antagonized (acts as an agonist). Modulation
can increase activity more than 1-fold, 2-fold, 3-fold, 5-fold,
10-fold, 100-fold, etc., over baseline values. Modulation can also
decrease its activity below baseline values.
[0075] As used herein, the term "selective for tumor cells" refers
to the effects of the Coenzyme Q10 pharmaceutical compositions,
such as inhibition of tumor growth, apoptosis, anti-angiogenic
effects and which are not detectable when applied to normal cells,
as described in detail in the examples which follow.
CoQ10 Compositions
[0076] In a preferred embodiment, the invention provides CoQ10
compositions for the treatment of cancer. Preferably, the
compositions comprise at least about 1% to about 25% CoQ10 w/w,
more preferably, between about 1% to about 20% CoQ10 w/w. In the
representative embodiment described in the Examples section below,
a topical formulation of CoQ10 is applied to the skin of a
tumor-bearing animal to reduce the growth rate of the tumor. CoQ10
can be obtained from Pure Prescriptions (San Diego, Calif.) in
powdered form in any suitable quantity (e.g., 1 kilogram). To
deliver a CoQ10-containing composition, any suitable carrier can be
used. Liposomes, for example, may be used as a carrier. An
exemplary liposomal formulation is composed of Phospholipon 90G
(American Lechitin, Stanford, Conn.), Phospholipon 9011 (American
Lechitin, Stanford, Conn.), Glycerol, Butylated hydroxytoluene
(BHT), Ethanol, Medium Chain Triglycerides (MCT), lavender
(Sigma-Aldrich, St. Louis, Mo.) and Coenzyme Q10 (Pure
Prescriptions, San Diego, Calif.). An example of a protocol for
preparing this formulation entails first dissolving 10 g of
Phospholipon 90H, 5 g Phospholipon 90G, with 1.5 g MCT, 0.3 g BHT,
and 9 ml of ethanol at 75.degree. C. Next, 12 g of Coenzyme Q10 are
dissolved into the mixture. 65 ml of 1 mM phosphate buffer (pH 8.2)
prepared with nitrogen saturated water, 13.3 g glycerol, and 50
.mu.L of lavender are added. The above mixture is blended in a
high-speed blender at 12,000 RPM to form a cream. The cream is
stored at 4.degree. C. until used.
Subjects
[0077] Because subjects from many different species have tumors and
are susceptible to acquiring a tumor, the invention is compatible
with many types of animal subjects. A non-exhaustive exemplary list
of such animals includes mammals such as mice, rats, rabbits,
goats, sheep, pigs, horses, cattle, dogs, cats, and primates such
as monkeys, apes, and human beings. Those animal subjects known to
suffer from a skin cancer tumor are preferred for use in the
invention. In particular, human patients suffering from a skin
cancer tumor or other tumors are suitable animal subjects for use
in the invention. By adapting the methods taught herein to other
methods known in medicine or veterinary science (e.g., adjusting
doses of administered substances according to the weight of the
subject animal), the compositions utilized in the invention can be
readily optimized for use in other animals.
Pharmaceutical Compositions and Administration to a Subject
[0078] In a preferred embodiment, the compositions comprising CoQ10
are administered topically. It is preferable to present the active
ingredient, i.e. CoQ10 as a pharmaceutical formulation. Exemplary
compositions are described in detail in the examples which follow.
The active ingredient may comprise, for topical administration,
from 0.001% to about 20% w/w, by weight of the formulation in the
final product., although it may comprise as much as 30% w/w,
preferably from about 1% to about 20% w/w of the formulation. The
topical formulations of the present invention, comprise an active
ingredient together with one or more acceptable carrier(s) therefor
and optionally any other therapeutic ingredients(s). The carriers)
must be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not deleterious to the
recipient thereof.
[0079] The composition of the invention can be administered to a
patient either by themselves, or in pharmaceutical compositions
where it is mixed with suitable carriers or excipient(s). In
treating a patient exhibiting a disorder of interest, a
therapeutically effective amount of a agent or agents such as these
is administered. A therapeutically effective dose refers to that
amount of the compound that results in amelioration of symptoms or
a prolongation of survival in a patient.
[0080] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized.
[0081] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating plasma concentration range that
includes the IC.sub.50 as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by
HPLC.
[0082] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. (See e.g. Fingl et al., in The Pharmacological Basis of
Therapeutics, 1975, Ch. 1 p. 1). It should be noted that the
attending physician would know how to and when to terminate,
interrupt, or adjust administration due to toxicity, or to organ
dysfunctions. Conversely, the attending physician would also know
to adjust treatment to higher levels if the clinical response were
not adequate (precluding toxicity). The magnitude of an
administrated dose in the management of the oncogenic disorder of
interest will vary with the severity of the condition to be treated
and to the route of administration. The severity of the condition
may, for example, be evaluated, in part, by standard prognostic
evaluation methods. Further, the dose and perhaps dose frequency,
will also vary according to the age, body weight, and response of
the individual patient. A program comparable to that discussed
above may be used in veterinary medicine.
[0083] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
Remington's Pharmaceutical Sciences, 18.sup.th ed., Mack Publishing
Co., Easton, Pa. (1990). Suitable routes may include oral, rectal,
transdermal, vaginal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections, just to name a few.
[0084] The compositions described above may be administered to a
subject in any suitable formulation. In addition to treatment of
cancer with topical formulations of CoQ10, in other aspects of the
invention CoQ10 might be delivered by other methods. For example,
CoQ10 might be formulated for parenteral delivery, e.g., for
subcutaneous, intravenous, intramuscular, or intratumoral
injection. Other methods of delivery, for example, liposomal
delivery or diffusion from a device impregnated with the
composition might be used. The compositions may be administered in
a single bolus, multiple injections, or by continuous infusion (for
example, intravenously or by peritoneal dialysis). For parenteral
administration, the compositions are preferably formulated in a
sterilized pyrogen-free form. Compositions of the invention can
also be administered in vitro to a cell (for example, to induce
apoptosis in a cancer cell in an in vitro culture) by simply adding
the composition to the fluid in which the cell is contained.
[0085] Depending on the specific conditions being treated, such
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
Remington's Pharmaceutical Sciences, 18.sup.th ed., Mack Publishing
Co., Easton, Pa. (1990). Suitable routes may include oral, rectal,
transdermal, vaginal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections, just to name a few.
[0086] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0087] Use of pharmaceutically acceptable carriers to formulate the
compounds herein disclosed for the practice of the invention into
dosages suitable for systemic administration is within the scope of
the invention. With proper choice of carrier and suitable
manufacturing practice, the compositions of the present invention,
in particular, those formulated as solutions, may be administered
parenterally, such as by intravenous injection. The compounds can
be formulated readily using pharmaceutically acceptable carriers
well known in the art into dosages suitable for oral
administration. Such carriers enable the compounds of the invention
to be formulated as tablets, pills, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
patient to be treated.
[0088] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes, then administered as described above. Liposomes are
spherical lipid bilayers with aqueous interiors. All molecules
present in an aqueous solution at the time of liposome formation
are incorporated into the aqueous interior. The liposomal contents
are both protected from the external microenvironment and, because
liposomes fuse with cell membranes, are efficiently delivered into
the cell cytoplasm. Additionally, due to their hydrophobicity,
small organic molecules may be directly administered
intracellularly.
[0089] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
See, for example, FIG. 14. Determination of the effective amounts
is well within the capability of those skilled in the art,
especially in light of the detailed disclosure provided herein. In
addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions. The pharmaceutical compositions of the present invention
may be manufactured in a manner that is itself known, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levitating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0090] Formulations suitable for topical administration include
liquid or semi-liquid preparations suitable for penetration through
the skin to the site of where treatment is required, such as
liniments, lotions, creams, ointments or pastes, and drops suitable
for administration to the eye, ear, or nose. Drops according to the
present invention may comprise sterile aqueous or oily solutions or
suspensions and may be prepared by dissolving the active ingredient
in a suitable aqueous solution of a bactericidal and/or fungicidal
agent and/or any other suitable preservative, and preferably
including a surface active agent. The resulting solution may then
be clarified and sterilized by filtration and transferred to the
container by an aseptic technique. Examples of bactericidal and
fungicidal agents suitable for inclusion in the drops are
phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride
(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for
the preparation of an oily solution include glycerol, diluted
alcohol and propylene glycol.
[0091] Lotions according to the present invention include those
suitable for application to the skin or eye. An eye lotion may
comprise a sterile aqueous solution optionally containing a
bactericide and may be prepared by methods similar to those for the
preparation of drops. Lotions or liniments for application to the
skin may also include an agent to hasten drying and to cool the
skin, such as an alcohol or acetone, and/or a moisturizer such as
glycerol or an oil such as castor oil or arachis oil.
[0092] Creams, ointments or pastes according to the present
invention are semi-solid formulations of the active ingredient for
external application. They may be made by mixing the active
ingredient in finely-divided or powdered form, alone or in solution
or suspension in an aqueous or non-aqueous fluid, with the aid of
suitable machinery, with a greasy or non-greasy basis. The basis
may comprise hydrocarbons such as hard, soft or liquid paraffin,
glycerol, beeswax, a metallic soap; a mucilage; an oil of natural
origin such as ahnond, corn, arachis, castor or olive oil; wool fat
or its derivatives, or a fatty acid such as stearic or oleic acid
together with an alcohol such as propylene glycol or macrogels. The
formulation may incorporate any suitable surface active agent such
as an anionic, cationic or non-ionic surface active such as
sorbitan esters or polyoxyethylene derivatives thereof. Suspending
agents such as natural gums, cellulose derivatives or inorganic
materials such as silicaceous silicas, and other ingredients such
as lanolin, may also be included.
[0093] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0094] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxy-methylcellulose, and/or polyvinyl pyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0095] Dragee cores are provided with suitable coating. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0096] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
[0097] The composition can include a buffer system, if desired.
Buffer systems are chosen to maintain or buffer the pH of
compositions within a desired range. The term "buffer system" or
"buffer" as used herein refers to a solute agent or agents which,
when in a water solution, stabilize such solution against a major
change in pH (or hydrogen ion concentration or activity) when acids
or bases are added thereto. Solute agent or agents which are thus
responsible for a resistance or change in pH from a starting
buffered pH value in the range indicated above are well known.
While there are countless suitable buffers, potassium phosphate
monohydrate is a preferred buffer.
[0098] The final pH value of the pharmaceutical composition may
vary within the physiological compatible range. Necessarily, the
final pH value is one not irritating to human skin and preferably
such that transdermal transport of the active compound, i.e. CoQ10
is facilitated. Without violating this constraint, the pH may be
selected to improve CoQ10 compound stability and to adjust
consistency when required. In one embodiment, the preferred pH
value is about 3.0 to about 7.4, more preferably about 3.0 to about
6.5, most preferably from about 3.5 to about 6.0.
[0099] For preferred topical delivery vehicles the remaining
component of the composition is water, which is necessarily
purified, e.g., deionized water. Such delivery vehicle compositions
contain water in the range of more than about 50 to about 95
percent, based on the total weight of the composition. The specific
amount of water present is not critical, however, being adjustable
to obtain the desired viscosity (usually about 50 cps to about
10,000 cps) and/or concentration of the other components. The
topical delivery vehicle preferably has a viscosity of at least
about 30 centipoises.
[0100] Other known transdermal skin penetration enhancers can also
be used to facilitate delivery of CoQ10. Illustrative are
sulfoxides such as dimethylsulfoxide (DMSO) and the like; cyclic
amides such as 1-dodecylazacycloheptane-2-one (Azone.TM., a
registered trademark of Nelson Research, Inc.) and the like; amides
such as N,N-dimethyl acetamide (DMA) N,N-diethyl toluamide,
N,N-dimethyl formamide, N,N-dimethyl octamide, N,N-dimethyl
decamide, and the like; pyrrolidone derivatives such as
N-methyl-2-pyrrolidone, 2-pyrrolidone, 2-pyrrolidone-5-carboxylic
acid, N-(2-hydroxyethyl)-2-pyrrolidone or fatty acid esters
thereof, 1-lauryl-4-methoxycarbonyl-2-pyrrolidone,
N-tallowalkylpyrrolidones, and the like; polyols such as propylene
glycol, ethylene glycol, polyethylene glycol, dipropylene glycol,
glycerol, hexanetriol, and the like; linear and branched fatty
acids such as oleic, linoleic, lauric, valeric, heptanoic, caproic,
myristic, isovaleric, neopentanoic, trimethyl hexanoic, isostearic,
and the like; alcohols such as ethanol, propanol, butanol, octanol,
oleyl, stearyl, linoleyl, and the like; anionic surfactants such as
sodium laurate, sodium lauryl sulfate, and the like; cationic
surfactants such as benzalkonium chloride, dodecyltrimethylammonium
chloride, cetyltrimethylammonium bromide, and the like; non-ionic
surfactants such as the propoxylated polyoxyethylene ethers, e.g.,
Poloxamer 231, Poloxamer 182, Poloxamer 184, and the like, the
ethoxylated fatty acids, e.g., Tween 20, Myrj 45, and the like, the
sorbitan derivatives, e.g., Tween 40, Tween 60, Tween 80, Span 60,
and the like, the ethoxylated alcohols, e.g., polyoxyethylene (4)
lauryl ether (Brij 30), polyoxyethylene (2) oleyl ether (Brij 93),
and the like, lecithin and lecithin derivatives, and the like; the
terpenes such as D-limonene, .alpha.-pinene, .beta.-carene,
.alpha.-terpineol, carvol, carvone, menthone, limonene oxide,
.alpha.-pinene oxide, eucalyptus oil, and the like.
[0101] Also suitable as skin penetration enhancers are organic
acids and esters such as salicyclic acid, methyl salicylate, citric
acid, succinic acid, and the like.
Angiogenesis and Angiogenesis-Dependent Diseases
[0102] As used herein, the terms "angiogenesis inhibitory",
"angiogenesis inhibiting" or "anti-angiogenic" include
vasculogenesis, and are intended to mean effecting a decrease in
the extent, amount, or rate of neovascularization. Effecting a
decrease in the extent, amount, or rate of endothelial cell
proliferation or migration in the tissue is a specific example of
inhibiting angiogenesis.
[0103] The term "angiogenesis inhibitory composition" refers to a
composition comprising CoQ10 which inhibits tumor mediated
angiogenesis processes such as endothelial cell migration,
proliferation, tube formation and subsequently leading to the
inhibition of the generation of new blood vessels from existing
ones, and consequently the inhibition of angiogenesis-dependent
diseases, for example, angiogenesis mediated by tumors. See, for
example FIGS. 29A and 29B wherein a composition comprising CoQ10
inhibits tumor-mediated angiogenesis in a tissue as compared to
control tissue in the absence of any CoQ10. The composition
comprising CoQ10 is described in detail in the examples which
follow.
[0104] As used herein, the term "angiogenesis-dependent disease" is
intended to mean a disease where the process of angiogenesis or
vasculogenesis sustains or augments a pathological condition. In
particular, angiogenesis-dependent disease refers to tumor-mediated
angiogenesis.
[0105] Angiogenesis is the formation of new blood vessels from
pre-existing capillaries or post-capillary venules. Vasculogenesis
results from the formation of new blood vessels arising from
angioblasts which are endothelial cell precursors. Both processes
result in new blood vessel formation and are included in the
meaning of the term angiogenesis-dependent diseases. Similarly, the
term "angiogenesis" as used herein is intended to include de novo
formation of vessels such as that arising from vasculogenesis as
well as those arising from branching and sprouting of existing
vessels, capillaries and venules.
[0106] Angiogenesis, including vasculogenesis, is an important
physiological process, without which embryonic development and
wound healing would not occur. However, angiogenesis is also
inappropriately recruited into numerous pathological conditions as
a means to provide adequate blood and nutrient supply to the cells
within the affected tissue. Many of these pathological conditions
involve aberrant cell proliferation or regulation. Such conditions
in which angiogenesis is believed to be important are referred to
herein as angiogenesis-dependent diseases. However, methods of the
invention also can be used beneficially to inhibit angiogenesis
associated with normal physiological processes. For example, the
inhibition of angiogenesis associated with the menstrual cycle can
be prophylactically used as an effective method of birth control.
Therefore, the description below in reference to the treatment of
angiogenesis-dependent diseases are also applicable to the
inhibition of normal angiogenic responses where a prophylactic or
therapeutic need or benefit exists.
[0107] Angiogenesis-dependent diseases include, for example,
inflammatory disorders such as immune and non-immune inflammation,
rheumatoid arthritis, chronic articular rheumatism and psoriasis;
disorders associated with inappropriate or inopportune invasion of
vessels such as diabetic retinopathy, neovascular glaucoma,
retinopathy of prematurity, macular degeneration, corneal graft
rejection, retrolental fibroplasia, rubeosis, capillary
proliferation in atherosclerotic plaques and osteoporosis; and
cancer associated disorders, including for example, solid tumors,
tumor metastases, blood born tumors such as leukemias,
angiofibromas, Kaposi sarcoma, benign tumors such as hemangiomas,
acoustic neuromas, neurofibromas, trachomas, and pyogenic
granulomas, as well as other cancers which require
neovascularization to support tumor growth. Additional examples of
angiogenesis-dependent diseases include, for example, Osler-Webber
Syndrome; myocardial angiogenesis; plaque neovascularization;
telangiectasia; hemophiliac joints and wound granulation. Other
diseases in which angiogenesis plays a role in the maintenance or
progression of the pathological state are known to those skilled in
the art and are similarly intended to be included within the
meaning of the term used herein. Preferably, angiogenesis-mediated
diseases refers to tumor induced angiogenesis.
In Vitro Biological Assay of Angiogenesis Inhibiting Activity
[0108] The CoQ10 compounds of the instant invention can be tested
for their angiogenesis inhibiting activity in several assay systems
in vitro and are well within the knowledge of one of ordinary skill
in the art. Endothelial cells, for example, human umbilical vein
endothelial cells (HMVEC) or human microvascular endothelial cells
(HMVEC) can be prepared or obtained commercially, are mixed at a
concentration of 2.times.10.sup.5 cells/mL with fibrinogen (5 mg/mL
in phosphate buffered saline (PBS) in a 1:1 (v/v) ratio. Thrombin
is added (5 units/mL final concentration) and the mixture
immediately transferred to a 24-well plate (0.5 mL per well). The
fibrin gel is allowed to form and then vascular endothelial growth
factor (VEGF) and fibroblast growth factor basic (FGF2) are added
to the wells (each at 5 ng/mL final concentration) along with the
test compound, as described in the Examples which follow. The cells
are incubated at 37.degree. C. in 5% CO.sub.2 for 4 days at which
time the cells in each well are counted and classified as either
rounded, elongated with no branches, elongated with one branch, or
elongated with 2 or more branches. Results are expressed as the
average of 5 different wells for each concentration of compound.
Typically, in the presence of angiogenic inhibitors, cells remain
either rounded or form undifferentiated tubes (e.g., 0 or 1
branch). This assay is recognized in the art to be predictive of
angiogenic efficacy (or angiogenesis inhibiting activity) in vivo
(Grant et al., In Vitro Cell Dev. Biol. 27A:327-336 (1991); Min et
al., Cancer Res. 56:2428-2433 (1996)).
[0109] In an alternate assay, endothelial cell tube formation is
observed when endothelial cells are cultured on Matrigel.TM.
matrix-coated plates, commercially available from Becton Dickinson
of Bedford, Pa. (Schnaper et al., J. Cell. Physiol. 165:107-118
(1995)). Endothelial cells (1.times.10.sup.4 cells/well) are
transferred onto Matrigel.TM. matrix-coated 24-well plates, and
tube formation is quantitated after 48 hours. Inhibitors are tested
by adding them either at the same time as the endothelial cells or
at various time points thereafter.
[0110] This assay models angiogenesis by presenting to the
endothelial cells a particular type of basement membrane, namely
the layer of matrix which migrating and differentiating endothelial
cells might be expected to first encounter. In addition to bound
growth factors, the matrix components found in Matrigel.TM. matrix
(and in basement membranes in situ) or proteolytic products thereof
may also be stimulatory for endothelial cell tube formation which
makes this model complementary to the fibrin gel angiogenesis
model.
[0111] Additionally, angiogenic activities of compounds of the
present invention can be evaluated by the chick chorioallantoic
membrane (CAM) assay (Oikawa et al., Cancer Lett. 59:57-66
(1991)).
Combination Therapies
[0112] The CoQ10 therapeutic compositions of the present invention
may be combined with any other methods generally employed in the
treatment of the particular tumor, disease or disorder that the
patient exhibits. So long as a particular therapeutic approach is
not known to be detrimental to the patient's condition in itself,
and does not significantly counteract the the CoQ10 composition
treatment, its combination with the present invention is
contemplated.
[0113] In connection solid tumor treatment, the present invention
may be used in combination with classical approaches, such as
surgery, radiotherapy, chemotherapy, and the like. The invention
therefore provides combined therapies in which the CoQ10
therapeutic compositions are used simultaneously with, before, or
after surgery or radiation treatment; or are administered to
patients with, before, or after conventional chemotherapeutic,
radiotherapeutic or other anti-angiogenic agents, or targeted
immunotoxins or coaguligands.
[0114] Combination therapy for other vascular diseases is also
contemplated. A particular example of such is benign prostatic
hyperplasia (BPH), which may be treated with CoQ10 compositions in
combination other treatments currently practiced in the art. For
example, targeting of immunotoxins to markers localized within BPH,
such as PSA.
[0115] When one or more agents are used in combination with the
CoQ10 compositions, there is no requirement for the combined
results to be additive of the effects observed when each treatment
is conducted separately. Although at least additive effects are
generally desirable, any increased anti-tumor effect above one of
the single therapies would be of benefit. Also, there is no
particular requirement for the combined treatment to exhibit
synergistic effects, although this is certainly possible and
advantageous.
[0116] To practice combined anti-tumor therapy, one would simply
administer to an animal a the CoQ 10 composition construct in
combination with another anti-cancer agent in a manner effective to
result in their combined anti-tumor actions within the animal. The
agents would therefore be provided in amounts effective and for
periods of time effective to result in their combined presence
within the tumor vasculature and their combined actions in the
tumor environment. To achieve this goal, the CoQ10 compositions and
other anti-cancer agents may be administered to the animal
simultaneously, either in a single composition, or as two distinct
compositions using different administration routes.
[0117] Alternatively, the CoQ10 composition mediated treatment may
precede, or follow, the a second anti-cancer agent treatment by,
e.g., intervals ranging from minutes to weeks. In certain
embodiments where the anti-cancer agent and the CoQ10 composition
are applied separately to the animal, one would ensure that a
significant period of time did not expire between the time of each
delivery, such that the anti-cancer agent and the CoQ10 composition
would still be able to exert an advantageously combined effect on
the tumor. In such instances, it is contemplated that one would
contact the tumor with both agents within about 5 minutes to about
one week of each other and, more preferably, within about 12-72
hours of each other, with a delay time of only about 12-48 hours
being most preferred.
[0118] The general use of combinations of substances in cancer
treatment is well known. For example, U.S. Pat. No. 5,710,134
(incorporated herein by reference) discloses components that induce
necrosis in tumors in combination with non-toxic substances or
"prodrugs". The enzymes set free by necrotic processes cleave the
non-toxic "prodrug" into the toxic "drug", which leads to tumor
cell death. Also, U.S. Pat. No. 5,747,469 (incorporated herein by
reference) discloses the combined use of viral vectors encoding p53
and DNA damaging agents. Any such similar approaches can be used
with the present invention.
[0119] In some situations, it may even be desirable to extend the
time period for treatment significantly, where several days (2, 3,
4, 5, 6 or 7), several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even
several months (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations. This would be advantageous in
circumstances where one treatment was intended to substantially
destroy the tumor, such as the CoQ10 composition treatment, and
another treatment was intended to prevent micrometastasis or tumor
re-growth, such as the administration of an anti-angiogenic
agent.
[0120] It also is envisioned that more than one administration of
either the CoQ10 composition or another anti-cancer agent will be
utilized. The CoQ10 composition and anti-cancer agents may be
administered interchangeably, on alternate days or weeks; or a
sequence of the CoQ10 composition treatment may be given, followed
by a sequence of anti-cancer agent therapy. In any event, to
achieve tumor regression using a combined therapy, all that is
required is to deliver both agents in a combined amount effective
to exert an anti-tumor effect, irrespective of the times for
administration.
[0121] In terms of surgery, any surgical intervention may be
practiced in combination with the present invention. In connection
with radiotherapy, any mechanism for inducing DNA damage locally
within tumor cells is contemplated, such as .gamma.-irradiation,
X-rays, UV-irradiation, microwaves and even electronic emissions
and the like. The directed delivery of radioisotopes to tumor cells
is also contemplated, and this may be used in connection with a
targeting antibody or other targeting means.
[0122] Cytokine therapy also has proven to be an effective partner
for combined therapeutic regimens. Various cytokines may be
employed in such combined approaches. Examples of cytokines include
IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-13, TGF-.beta., GM-CSF, M-CSF, G-CSF,
TNF.alpha., TNF.beta., LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF,
OSM, TMF, PDGF, IFN-.alpha., IFN-.beta., IFN-.gamma.. Cytokines are
administered according to standard regimens, consistent with
clinical indications such as the condition of the patient and
relative toxicity of the cytokine. Uteroglobins may also be used to
prevent or inhibit metastases (U.S. Pat. No. 5,696,092;
incorporated herein by reference).
CoQ10 Compositions and Combination Chemotherapeutics
[0123] In certain embodiments, the CoQ10 composition of the present
invention may be administered in combination with another
chemotherapeutic agent. Irrespective of the underlying
mechanism(s), a variety of chemotherapeutic agents may be used in
the combined treatment methods disclosed herein. Therapeutic agents
can include, for example, chemotherapeutic agents such as,
cyclophosphamide (CTX, 25 mg/kg/day, p.o.), taxanes (paclitaxel or
docetaxel), busulfan, cisplatin, methotrexate, daunorubicin,
doxorubicin, melphalan, cladribine, vincristine, vinblastine,
chlorambucil, tamoxifen, taxol, etoposide (VP-16), adriamycin,
5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycin C,
cisplatin (CDDP), combretastatin(s) and derivatives and prodrugs
thereof.
[0124] As will be understood by those of ordinary skill in the art,
the appropriate doses of chemotherapeutic agents will be generally
around those already employed in clinical therapies wherein the
chemotherapeutics are administered alone or in combination with
other chemotherapeutics. By way of example only, agents such as
cisplatin, and other DNA alkylating may be used. Cisplatin has been
widely used to treat cancer, with efficacious doses used in
clinical applications of 20 mg/m.sup.2 for 5 days every three weeks
for a total of three courses. Cisplatin is not absorbed orally and
must therefore be delivered via injection intravenously,
subcutaneously, intratumorally or intraperitoneally.
[0125] Further useful agents include compounds that interfere with
DNA replication, mitosis and chromosomal segregation. Such
chemotherapeutic compounds include adriamycin, also known as
doxorubicin, etoposide, verapamil, podophyllotoxin, and the like.
Widely used in a clinical setting for the treatment of neoplasms,
these compounds are administered through bolus injections
intravenously at doses ranging from 25-75 mg/m.sup.2 at 21 day
intervals for adriamycin, to 35-50 mg/m.sup.2 for etoposide
intravenously or double the intravenous dose orally.
[0126] Agents that disrupt the synthesis and fidelity of
polynucleotide precursors may also be used. Particularly useful are
agents that have undergone extensive testing and are readily
available. As such, agents such as 5-fluorouracil (5-FU) are
preferentially used by neoplastic tissue, making this agent
particularly useful for targeting to neoplastic cells. Although
quite toxic, 5-FU, is applicable in a wide range of carriers,
including topical, however intravenous administration with doses
ranging from 3 to 15 mg/kg/day being commonly used.
[0127] The skilled artisan is directed to "Remington's
Pharmaceutical Sciences" 15th Edition, chapter 33, in particular
pages 624-652, for non-limiting examples of other chemotherapeutic
agents that can be used in combination therapies with the CoQ10
compositions. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The
physician responsible for administration will be able to determine
the appropriate dose for the individual subject.
Anti-Angiogenics
[0128] The term "angiogenesis" refers to the generation of new
blood vessels, generally into a tissue or organ. Under normal
physiological conditions, humans or animals undergo angiogenesis
only in very specific restricted situations. For example,
angiogenesis is normally observed in wound healing, fetal and
embryonic development and formation of the corpus luteum,
endometrium and placenta. Uncontrolled (persistent and/or
unregulated) angiogenesis is related to various disease states, and
occurs during tumor growth and metastasis.
[0129] Both controlled and uncontrolled angiogenesis are thought to
proceed in a similar manner. Endothelial cells and pericytes,
surrounded by a basement membrane, form capillary blood vessels.
Angiogenesis begins with the erosion of the basement membrane by
enzymes released by endothelial cells and leukocytes. The
endothelial cells, which line the lumen of blood vessels, then
protrude through the basement membrane. Angiogenic stimulants
induce the endothelial cells to migrate through the eroded basement
membrane. The migrating cells form a "sprout" off the parent blood
vessel, where the endothelial cells undergo mitosis and
proliferate. The endothelial sprouts merge with each other to form
capillary loops, creating the new blood vessel.
[0130] As persistent, unregulated angiogenesis occurs during tumor
development and metastasis, the treatment methods of this invention
may be used in combination with any one or more "anti-angiogenic"
therapies. Exemplary anti-angiogenic agents that are useful in
connection with combined therapy are listed in Table 1. Each of the
agents listed therein is exemplary and by no means limiting.
TABLE-US-00001 TABLE 1 Inhibitors and Negative Regulators of
Angiogenesis Substances Angiostatin Endostatin 16 kDa prolactin
fragment Laminin peptides Fibronectin peptides Tissue
metalloproteinaseinhibitors (TIMP 1, 2, 3, 4) Plasminogen activator
inhibitors (PAI-1, -2) Tumor necrosis factor alpha (high dose, in
vitro) TGF-.beta.1 Interferons (IFN-.alpha., -.beta., .gamma.)
ELR-CXC Chemokines:IL-12; SDF-1; MIG;Platelet factor 4 (PF-4);
IP-10 Thrombospondin (TSP) SPARC 2-Methoxyoestradiol
Proliferin-related protein Suramin Thalidomide Cortisone Fumagillin
(AGM-1470; TNP-470) Tamoxifen Korean mistletoe extract (Viscum
album coloratum) Retinoids CM101 Dexamethasone Leukemia inhibitory
factor (LIF)
[0131] A certain preferred component for use in inhibiting
angiogenesis is a protein named "angiostatin". This component is
disclosed in U.S. Pat. Nos. 5,776,704; 5,639,725 and 5,733,876,
each incorporated herein by reference. Angiostatin is a protein
having a molecular weight of between about 38 kD and about 45 kD,
as determined by reducing polyacrylamide gel electrophoresis, which
contains approximately Kringle regions 1 through 4 of a plasminogen
molecule. Angiostatin generally has an amino acid sequence
substantially similar to that of a fragment of murine plasminogen
beginning at amino acid number 98 of an intact murine plasminogen
molecule.
[0132] The amino acid sequence of angiostatin varies slightly
between species. For example, in human angiostatin, the amino acid
sequence is substantially similar to the sequence of the above
described murine plasminogen fragment, although an active human
angiostatin sequence may start at either amino acid number 97 or 99
of an intact human plasminogen amino acid sequence. Further, human
plasminogen may be used, as it has similar anti-angiogenic
activity, as shown in a mouse tumor model.
[0133] Certain anti-angiogenic therapies have already been shown to
cause tumor regressions, and angiostatin is one such agent.
Endostatin, a 20 kDa COOH-terminal fragment of collagen XVIII, the
bacterial polysaccharide CM101, and the antibody LM609 also have
angiostatic activity. However, in light of their other properties,
they are referred to as anti-vascular therapies or tumor vessel
toxins, as they not only inhibit angiogenesis but also initiate the
destruction of tumor vessels through mostly undefined mechanisms.
Their combination with the present invention is clearly
envisioned.
[0134] Angiostatin and endostatin have become the focus of intense
study, as they are the first angiogenesis inhibitors that have
demonstrated the ability to not only inhibit tumor growth but also
cause tumor regressions in mice. There are multiple proteases that
have been shown to produce angiostatin from plasminogen including
elastase, macrophage metalloelastase (MME), matrilysin (MMP-7), and
92 kDa gelatinase B/type IV collagenase (MMP-9).
[0135] MME can produce angiostatin from plasminogen in tumors and
granulocyte-macrophage colony-stimulating factor (GMCSF)
upregulates the expression of MME by macrophages inducing the
production of angiostatin. The role of MME in angiostatin
generation is supported by the finding that MME is in fact
expressed in clinical samples of hepatocellular carcinomas from
patients. Another protease thought to be capable of producing
angiostatin is stromelysin-1 (MMP-3). MMP-3 has been shown to
produce angiostatin-like fragments from plasminogen in vitro.
[0136] CM101 is a bacterial polysaccharide that has been well
characterized in its ability to induce neovascular inflammation in
tumors. CM101 binds to and cross-links receptors expressed on
dedifferentiated endothelium that stimulates the activation of the
complement system. It also initiates a cytokine-driven inflammatory
response that selectively targets the tumor. It is an
antipathoangiogenic agent that downregulates the expression VEGF
and its receptors.
[0137] Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also
be used in combination with the present invention. These are both
angiogenesis inhibitors that associate with heparin and are found
in platelet .alpha.-granules. TSP-1 is a large 450 kDa multi-domain
glycoprotein that is constituent of the extracellular matrix. TSP-1
binds to many of the proteoglycan molecules found in the
extracellular matrix including, HSPGs, fibronectin, laminin, and
different types of collagen. TSP-1 inhibits endothelial cell
migration and proliferation in vitro and angiogenesis in vivo.
TSP-1 can also suppress the malignant phenotype and tumorigenesis
of transformed endothelial cells. The tumor suppressor gene p53 has
been shown to directly regulate the expression of TSP-1 such that,
loss of p53 activity causes a dramatic reduction in TSP-1
production and a concomitant increase in tumor initiated
angiogenesis.
[0138] PF4 is a 70aa protein that is member of the CXC ELR-family
of chemokines that is able to potently inhibit endothelial cell
proliferation in vitro and angiogenesis in vivo. PF4 administered
intratumorally or delivered by an adenoviral vector is able to
cause an inhibition of tumor growth.
[0139] Interferons and metalloproteinase inhibitors are two other
classes of naturally occurring angiogenic inhibitors that can be
combined with the present invention. The anti-endothelial activity
of the interferons has been known since the early 1980s, however,
the mechanism of inhibition is still unclear. It is known that they
can inhibit endothelial cell migration and that they do have some
anti-angiogenic activity in vivo that is possibly mediated by an
ability to inhibit the production of angiogenic promoters by tumor
cells. Vascular tumors in particular are sensitive to interferon,
for example, proliferating hemangiomas can be treated with
IFN.alpha..
[0140] Tissue inhibitors of metalloproteinases (TIMPs) are a family
of naturally occurring inhibitors of matrix metalloproteases (MMPs)
that can also inhibit angiogenesis and can be used in combined
treatment protocols with the present invention. MMPs play a key
role in the angiogenic process as they degrade the matrix through
which endothelial cells and fibroblasts migrate when extending or
remodeling the vascular network. In fact, one member of the MMPs,
MMP-2, has been shown to associate with activated endothelium
through the integrin .alpha.v.beta.3 presumably for this purpose.
If this interaction is disrupted by a fragment of MMP-2, then
angiogenesis is downregulated and in tumors growth is
inhibited.
[0141] There are a number of pharmacological agents that inhibit
angiogenesis, any one or more of which may be used in combination
with the present invention. These include AGM-1470/TNP-470,
thalidomide, and carboxyamidotriazole (CAI). Fumagillin was found
to be a potent inhibitor of angiogenesis in 1990, and since then
the synthetic analogues of fumagillin, AGM-1470 and TNP-470 have
been developed. Both of these drugs inhibit endothelial cell
proliferation in vitro and angiogenesis in vivo. TNP-470 has been
studied extensively in human clinical trials with data suggesting
that long-term administration is optimal.
[0142] Thalidomide was originally used as a sedative but was found
to be a potent teratogen and was discontinued. In 1994 it was found
that thalidomide is an angiogenesis inhibitor. Thalidomide is
currently in clinical trials as an anti-cancer agent as well as a
treatment of vascular eye diseases.
[0143] CAI is a small molecular weight synthetic inhibitor of
angiogenesis that acts as a calcium channel blocker that prevents
actin reorganization, endothelial cell migration and spreading on
collagen IV. CAI inhibits neovascularization at physiological
attainable concentrations and is well tolerated orally by cancer
patients. Clinical trials with CAI have yielded disease
stabilization in 49% of cancer patients having progressive disease
before treatment.
[0144] Cortisone in the presence of heparin or heparin fragments
was shown to inhibit tumor growth in mice by blocking endothelial
cell proliferation. The mechanism involved in the additive
inhibitory effect of the steroid and heparin is unclear although it
is thought that the heparin may increase the uptake of the steroid
by endothelial cells. The mixture has been shown to increase the
dissolution of the basement membrane underneath newly formed
capillaries and this is also a possible explanation for the
additive angiostatic effect. Heparin-cortisol conjugates also have
potent angiostatic and anti-tumor effects activity in vivo.
[0145] Further specific angiogenesis inhibitors, including, but not
limited to, Anti-Invasive Factor, retinoic acids and paclitaxel
(U.S. Pat. No. 5,716,981; incorporated herein by reference);
AGM-1470 (Ingber et al., Nature, 48:555-557 1990; incorporated
herein by reference); shark cartilage extract (U.S. Pat. No.
5,618,925; incorporated herein by reference); anionic polyamide or
polyurea oligomers (U.S. Pat. No. 5,593,664; incorporated herein by
reference); oxindole derivatives (U.S. Pat. No. 5,576,330;
incorporated herein by reference); estradiol derivatives (U.S. Pat.
No. 5,504,074; incorporated herein by reference); and
thiazolopyrimidine derivatives (U.S. Pat. No. 5,599,813;
incorporated herein by reference) are also contemplated for use as
anti-angiogenic compositions for the combined uses of the present
invention.
[0146] Compositions comprising an antagonist of an
.alpha..sub.v.beta..sub.3 integrin may also be used to inhibit
angiogenesis in combination with the present invention. As
disclosed in U.S. Pat. No. 5,766,591 (incorporated herein by
reference), RGD-containing polypeptides and salts thereof,
including cyclic polypeptides, are suitable examples of
.alpha..sub.v.beta..sub.3 integrin antagonists.
[0147] The antibody LM609 against the .alpha..sub.v.beta..sub.3
integrin also induces tumor regressions. Integrin
.alpha..sub.v.beta..sub.3 antagonists, such as LM609, induce
apoptosis of angiogenic endothelial cells leaving the quiescent
blood vessels unaffected. LM609 or other .alpha..sub.v.beta..sub.3
antagonists may also work by inhibiting the interaction of
.alpha..sub.v.beta..sub.3 and MMP-2, a proteolytic enzyme thought
to play an important role in migration of endothelial cells and
fibroblasts.
[0148] Apoptosis of the angiogenic endothelium in this case may
have a cascade effect on the rest of the vascular network.
Inhibiting the tumor vascular network from completely responding to
the tumor's signal to expand may, in fact, initiate the partial or
full collapse of the network resulting in tumor cell death and loss
of tumor volume. It is possible that endostatin and angiostatin
function in a similar fashion. The fact that LM609 does not affect
quiescent vessels but is able to cause tumor regressions suggests
strongly that not all blood vessels in a tumor need to be targeted
for treatment in order to obtain an anti-tumor effect.
[0149] Non-targeted angiopoietins, such as angiopoietin-2, may also
be used in combination with the present invention. The angiogenic
effects of various regulators involve an autocrine loop connected
with angiopoietin-2. The use of angiopoietin-2, angiopoietin-1,
angiopoietin-3 and angiopoietin-4, is thus contemplated in
conjunction with the present invention. Other methods of
therapeutic intervention based upon altering signaling through the
Tie2 receptor can also be used in combination herewith, such as
using a soluble Tie2 receptor capable of blocking Tie2 activation
(Lin et al., Proc. Natl. Acad. Sci., USA, 95(15):8829-34, 1998).
Delivery of such a construct using recombinant adenoviral gene
therapy has been shown to be effective in treating cancer and
reducing metastases (Lin et al., 1998).
CoQ10 Compositions and Combination Therapy with Apoptosis-Inducing
Agents
[0150] The CoQ10 composition treatment may also be combined with
treatment methods that induce apoptosis in any cells within the
tumor, including tumor cells and tumor vascular endothelial cells.
Although many anti-cancer agents may have, as part of their
mechanism of action, an apoptosis-inducing effect, certain agents
have been discovered, designed or selected with this as a primary
mechanism, as described below.
[0151] A number of oncogenes have been described that inhibit
apoptosis, or programmed cell death. Exemplary oncogenes in this
category include, but are not limited to, bcr-abl, bcl-2 (distinct
from bcl-1, cyclin Dl; GenBank accession numbers M14745, X06487;
U.S. Pat. Nos. 5,650,491; and 5,539,094; each incorporated herein
by reference) and family members including Bcl-xl, Mcl-1, Bak, A1,
A20. Overexpression of bcl-2 was first discovered in T cell
lymphomas. bcl-2 functions as an oncogene by binding and
inactivating Bax, a protein in the apoptotic pathway. Inhibition of
bcl-2 function prevents inactivation of Bax, and allows the
apoptotic pathway to proceed. Thus, inhibition of this class of
oncogenes, e.g., using antisense nucleotide sequences, is
contemplated for use in the present invention in aspects wherein
enhancement of apoptosis is desired (U.S. Pat. Nos. 5,650,491;
5,539,094; and 5,583,034; each incorporated herein by
reference).
[0152] Many forms of cancer have reports of mutations in tumor
suppressor genes, such as p53. Inactivation of p53 results in a
failure to promote apoptosis. With this failure, cancer cells
progress in tumorigenesis, rather than become destined for cell
death. Thus, provision of tumor suppressors is also contemplated
for use in the present invention to stimulate cell death. Exemplary
tumor suppressors include, but are not limited to, p53,
Retinoblastoma gene (Rb), Wilm's tumor (WT1), bax alpha,
interleukin-1.beta.-converting enzyme and family, MEN-1 gene,
neurofibromatosis, type 1 (NF1), cdk inhibitor p16, colorectal
cancer gene (DCC), familial adenomatosis polyposis gene (FAP),
multiple tumor suppressor gene (MTS-1), BRCA1 and BRCA2.
[0153] Preferred for use are the p53 (U.S. Pat. Nos. 5,747,469;
5,677,178; and 5,756,455; each incorporated herein by reference),
Retinoblastoma, BRCA1 (U.S. Pat. Nos. 5,750,400; 5,654,155;
5,710,001; 5,756,294; 5,709,999; 5,693,473; 5,753,441; 5,622,829;
and 5,747,282; each incorporated herein by reference), MEN-1
(GenBank accession number U93236) and adenovirus E1A (U.S. Pat. No.
5,776,743; incorporated herein by reference) genes.
[0154] Other compositions that may be used include genes encoding
the tumor necrosis factor related apoptosis inducing ligand termed
TRAIL, and the TRAIL polypeptide (U.S. Pat. No. 5,763,223;
incorporated herein by reference); the 24 kD apoptosis-associated
protease of U.S. Pat. No. 5,605,826 (incorporated herein by
reference); Fas-associated factor 1, FAF1 (U.S. Pat. No. 5,750,653;
incorporated herein by reference). Also contemplated for use in
these aspects of the present invention is the provision of
interleukin-1.beta.-converting enzyme and family members, which are
also reported to stimulate apoptosis.
[0155] Compounds such as carbostyril derivatives (U.S. Pat. Nos.
5,672,603; and 5,464,833; each incorporated herein by reference);
branched apogenic peptides (U.S. Pat. No. 5,591,717; incorporated
herein by reference); phosphotyrosine inhibitors and
non-hydrolyzable phosphotyrosine analogs (U.S. Pat. Nos. 5,565,491;
and 5,693,627; each incorporated herein by reference); agonists of
RXR retinoid receptors (U.S. Pat. No. 5,399,586; incorporated
herein by reference); and even antioxidants (U.S. Pat. No.
5,571,523; incorporated herein by reference) may also be used.
Tyrosine kinase inhibitors, such as genistein, may also be linked
to ligands that target a cell surface receptor (U.S. Pat. No.
5,587,459; incorporated herein by reference).
Effective Amounts
[0156] The compositions described above are preferably administered
to a subject in an effective amount. An effective amount is an
amount which is capable of producing a desirable result in a
treated animal or cell (for example, to induce apoptosis or impair
mitosis in a cell in the animal or a culture). As is well known in
the medical and veterinary arts, dosage for any one animal depends
on many factors, including the particular animal's size, body
surface area, age, the particular composition to be administered,
time and route of administration, general health, and other drugs
being administered concurrently. It is expected that an appropriate
dosage for topical administration of the compositions of the
invention would be in the range of about 1.5-4.0 mg CoQ10/kg of
body weight (e.g., 200 mg for subjects ranging from 110 to 300
lbs). An effective amount for use with a cell in culture will also
vary, but can be readily determined empirically (for example, by
adding varying concentrations to the cell and selecting the
concentration that best produces the desired result). It is
expected that an appropriate concentration would be in the range of
about 5-200 .mu.M.
Method for Inhibiting Cancer Cell Growth
[0157] The invention provides a method for inhibiting tumor cell
growth or increasing the rate of tumor cell apoptosis. The method
includes the steps of contacting a tumor cell with a composition
including a sufficient amount of CoQ10 to kill or at least retard
mitosis in the tumor cell. The method may be used to inhibit the
growth of numerous types of cancerous tumor cells. Coenzyme Q10 has
been tested and shown to be effective against melanoma, squamous,
and breast cancer cells. Coenzyme Q10 is expected to be effective
against other cancers as well, particularly those derived from
epithelial, mesenchymal, and hemopoietic origins.
[0158] Any suitable formulation of CoQ10 can be used in methods of
the invention. Typical formulations are topical liposomal
formulations of coenzyme Q10 of varying concentrations. In addition
to topical administration, CoQ10-containing formulations can be
administered to a subject via injection (e.g., IP, IV, IM, SQ).
[0159] In a method of reducing the rate of tumor cell growth or
increasing the rate of tumor cell apoptosis in vitro, CoQ10 is
dissolved in 2-propanol followed by dilution in a desired medium
(as described in example 1 below). In an in vivo method of reducing
the rate of tumor cell growth or increasing the rate of tumor cell
apoptosis, a CoQ10-containing cream is applied topically daily to
the target site until tumor regression occurs (as described in
examples 2 and 3). In another in vivo method, a CoQ10-containing
formulation is administered to a subject via injection (e.g., IP,
IV, IM, SQ).
[0160] Inhibition of tumor cell growth manifested by administration
of the CoQ10 compositions described herein, that is compositions
comprising about 1% to about 25% coenzyme Q10, refers to one or
more of the following effects: (1) inhibition, to some extent, of
tumor growth, including, (i) slowing down and (ii) complete growth
arrest; (2) reduction in the number of tumor cells; (3) maintaining
tumor size; (4) reduction in tumor size; (5) inhibition, including
(i) reduction, (ii) slowing down or (iii) complete prevention, of
tumor cell infiltration into peripheral organs; (6) inhibition,
including (i) reduction, (ii) slowing down or (iii) complete
prevention, of metastasis; (7) enhancement of anti-tumor immune
response, which may result in (i) maintaining tumor size, (ii)
reducing tumor size, (iii) slowing the growth of a tumor, (iv)
reducing, slowing or preventing invasion and/or (8) relief, to some
extent, of the severity or number of one or more symptoms
associated with the disorder.
[0161] In preferred embodiments, administration of coenzyme Q10
compositions results in one or more phenotypes of a tumor cell
being inhibited. For example, inhibition of tumor growth, reduction
of tumor size, inhibition of metastasis, reduction in the number of
tumor cells and the like. Each of these phenotypes of a tumor cell
can be measured using standard assays, such as for example,
imaging, mechanical measurements, in vitro assays and the like.
Kits and Formulations
[0162] The invention also provides a kit for reducing the rate of
tumor growth in a subject. The kit of the invention includes a
composition comprising CoQ10 and a pharmaceutically acceptable
carrier as well as printed instructions for using the composition
to reduce the rate of tumor growth in a subject.
[0163] Active components can be present in solid, semi-solid or
liquid form. Solid forms include for example, powders, granules and
flakes. Semi-solid forms include, for example, gels, creams,
gelatins and ointments. These and other active agents embraced by
the present invention are known to those of ordinary skill in the
art and, in most cases, are commercially available from suppliers
such as Compound Solutions, Inc., Escondido, Calif. Information on
these and other active and inactive agents embraced by the
invention, and their commercial suppliers is available from various
trade manuals, most particularly, Remington's Pharmaceutical
Sciences, United States Pharmacopoeia (USP), National Formulary
(NF), Merck Index, Physician's Desk Reference (PDR) and Chemical
Abstracts.
[0164] The kits of the invention will also generally contain at
least one inactive agent. As used herein, inactive agents are
agents which do not provide any therapeutic benefit to the subject
to whom they are administered. Instead, inactive agents can
function in many other ways such as to provide a base in which the
active agent can be dissolved or suspended, to dilute the active
agent in order to provide proper doses upon administration, to
facilitate the dissolution or suspension of the active agent, or to
prevent oxidation of the active agent by removing air bubbles from
the final compounded suspension. In some embodiments of the
invention, the kits lack an inactive agent, and rather contain two
or more active agents.
[0165] Base agents such as creams, oils, gels or ointments are
suitable for topical or suppository applications. The choice of
suitable inactive base agent for use in the kits of the invention
will depend upon the active agent to be compounded. Suitable base
agents will be known to the ordinary artisan. Alternatively,
Remington's Pharmaceutical Sciences, the Physician Desk Reference
(PDR) or other manuals as listed above, can be consulted in making
this determination.
[0166] Examples of inactive base agents or components include, for
example, lanolin, hydrophilic ointment, white ointment, yellow
ointment, polyethylene glycol ointment, petrolatum, hydrophilic
petrolatum, white petrolatum, rose water ointment, squalene,
hydrogenated vegetable oil (Type II), ultrasound gel, pluronic
lecithin organogel (PLO) gel, cream.
[0167] The term "petrolatum" as used herein means petrolatum
ointment, petrolatum gel or petrolatum cream, all of which are
commercially available. It is well within the realm of the ordinary
pharmaceutical artisan to determine which form of petrolatum is
most appropriate for a specific kit.
[0168] A commercially available ultrasound base is either
POLYSONIC.TM. (ultrasound gel) ultrasound lotion or Aquasonic
ultrasound 100 gel manufactured by Parker Laboratories, Inc.
(Fairfield, N.J.) or EcoGel 100 or EcoGel 200 manufactured by
Eco-Med (Mississauga, Ontario, Canada), the compositions of which
may include cetyl alcohol, liquid paraffin, polymer, surfactants,
preservatives such as propyl paraben and methyl paraben in
bacteriostatic concentration, fragrance, and reverse osmosis water.
As used herein, a gel is a base with a higher viscosity than a
lotion. The physical characteristics of the POLYSONIC.TM.
(ultrasound gel) ultrasound lotion and the EcoGel 100 include pH
range of 6.5-7.0, density of 1.04 g/cm.sup.3, viscosity of 35,000
to 70,000 cps and acoustic impedence of 1.60 (10.sup.5 g/cm.sup.2
sec). The physical characteristics of Aquasonic ultrasound 100 gel
or EcoGel 200 are similar to those of POLYSONIC.TM. (ultrasound
gel) ultrasound lotion and EcoGel 100 except that their viscosity
is 80,000 to 110,000 cps. These lotions and gels are available in a
clear, colorless form or in a blue colored form.
[0169] Liquid bases are recommended for orally administered
pharmaceuticals. In some embodiments of the invention, at least one
active agent, e.g. CoQ10, will be supplied already co-mingled with
an inactive agent. Examples of this include the combination of
magnesium hydroxide and aluminum hydroxide (commercially available
as MAALOX.TM. (magnesium hydroxide/aluminum hydroxide)), and
diphenhydramine HCl (commercially available as BENADRYL.TM.
(diphenhydramine hydrochloride)). Both MAALOX.TM. (magnesium
hydroxide/aluminum hydroxide) and BENADRYL.TM. (diphenhydramine
hydrochloride) are supplied by their respective manufacturers as a
combination of active and inactive agents.
[0170] Sterile base solutions are preferred for parenteral (i.e.,
injection), aerosol (i.e., inhalation) and ophthalmic routes of
administration. The administration may, for example, be
intravenous, intraperitoneal, intramuscular, intracavity,
subcutaneous or transdermal. Preparations for parenteral
administration includes sterile aqueous or nonaqueous solutions,
suspensions and emulsions. The compounded pharmaceuticals,
preferably those intended for parenteral, inhalation or ophthalmic
routes of administration, may be prepared and administered in
inactive agents which are pharmaceutically-acceptable. As used
herein, a pharmaceutically-acceptable carrier means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the active agents and that is compatible
with the biological systems such of a tissue or organism. The
physiologically acceptable carrier must be sterile for in vivo
administration. Pharmaceutically acceptable carriers include
diluents, fillers, salts, buffers, stabilizers, solubilizers and
other materials which are well-known in the art. The
characteristics of the carrier will depend on the route of
administration. In general, pharmaceutically-acceptable agents or
carriers are well-known to those of ordinary skill in the art. In
some embodiments, suitable sterile solutions include albuterol and
ipratropium inhalation solution; papaverine, phentolamine and
prostaglandin injection solution; fentanyl citrate injection
solution and cyclosporine ophthalmic drops.
[0171] Examples of nonaqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil such as olive oil, an injectable
organic esters such as ethyloliate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers, (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
antioxidants, chelating agents, and inert gases and the like. Those
of skill in the art can readily determine the various parameters
for preparing these alternative pharmaceutical compositions without
resort to undue experimentation.
[0172] Inactive agents may also include components which function
to preserve the integrity of the compounded formulation. This
latter category of inactive agents includes, for example,
anti-foaming agents. Anti-foaming agents are agents which function
to remove unwanted air trapped in a composition, perhaps during
mixing or agitation. The use of anti-foaming components is
particularly useful in the preparation of pharmaceuticals to be
used for ultrasound imaging due to the impedance of signal
transmission by air bubbles. Examples of other anti-foaming agents
useful in the compositions of the invention include
bisphenylhexamethicone, dimethicone, dimethiconol,
hexamethyldisiloxane, hexyl alcohol, isopropyl alcohol, petroleum
distillates, phenethyl disiloxane, phenyl trimethicone,
polysilicone-7, propyl alcohol, silica dimethyl silylate, silica
silylate, tetramethyl decynediol and trimethylsiloxysilicate. A
preferred anti-foaming agent is simethicone. Simethicone is a
mixture of about 90% dimethicone and 10% silicone dioxide (w/w).
Simethicone is used extensively as an anti-gas agent in
pharmaceutical products such as GAS-X.TM. (simethicone), MAALOX.TM.
(magnesium hydroxide/aluminum hydroxide), MYLANTA.TM. (aluminum,
magnesium simethicone), PHAZYME.TM. (simethicone), GENAZYME.TM.
(simethicone), and MYLICON.TM. (simethicone) Drops. Simethicone may
be used as an anti-foaming agent in any of the formulations
embraced by the invention.
[0173] Other inactive agents which can be included in the
formulations of the invention include stabilizers such as citric
acid, anti-oxidants such as sodium metabisulfite and preservatives
such as methyl or propyl paraben.
[0174] Another class of inactive agents is suspending agents.
Suspending agents are agents which facilitate the suspension and in
some cases the dissolution of an active agent in a base. Generally,
suspending agents ensure more uniform mixing of active and base
components. In order to administer a more uniform dose of a
compounded pharmaceutical to a patient, the compounded components
must be properly and homogeneously combined. If the active agent is
present as a powder, a uniform dispersion is sometimes difficult to
achieve using the traditional form of compounding.
[0175] A subcategory of suspending agents are solubilizers.
Solubilizers are agents which facilitate the dissolution of a solid
or, in some cases, a semi-solid agent in a base inactive agent. In
some embodiments of the invention, a solid-form active agent may be
dissolved in a suspending agent, prior to mixing it with the base
agent. Conversely, the suspending agent and the base agent may be
prepackaged together, particularly if the concern is ensuring the
uniform blending of active agent within the base component rather
than the loss of solid (i.e., powdery) active agent. In still other
variations, the suspending agent may be premixed with the base
inactive agent.
[0176] Suitable suspending agents useful in the compositions of the
invention include, but are not limited to, glycerin, hexylene
glycol, propylene glycol, sorbitol, acacia, cholesterol,
diethanolamine (adjunct), glyceryl monostearate, lanolin alcohols,
lecithin, mono- and di-glycerides, monoethanolamine (adjunct),
oleic acid (adjunct), oleyl alcohol (stabilizer), poloxamer,
polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 40
hydrogenated castor oil, polyoxyl 10 oleyl ether, polyoxyl 20
cetostearyl ether, polyoxyl 40 stearate, polysorbate 20,
polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol
diacetate, propylene glycol monostearate, sodium lauryl sulfate,
sodium stearate, sorbitan monolaurate, sorbitan monooleate,
sorbitan monopalmitate, sorbitan monstearate, stearic acid,
trolamine, emulsifying wax, benzalkonium chloride, benzethonium
chloride, cetylpyridinium chloride, docusate sodium, nonoxynol 9,
nonoxynol 10, octoxynol 9, polyoxyl 50 stearate, and tyloxapol.
[0177] Still other suspending agents include humectants and wetting
agents. Humectants are agents which retain moisture. Examples of
humectants include but are not limited to glycerin, hexylene
glycol, propylene glycol and sorbitol. The amounts of base and
non-base inactive agents will also depend upon the particular
compounded pharmaceutical to be made. Base agents can be provided
in quantities corresponding to final compounded preparations which
contain 0.5% to 99.99% of base agent, either in weight or in
volume. In preferred embodiments, the final concentration of the
base agent is 20%-80%. In even more preferred embodiments, the
final concentration of the base agent is 40%-80%.
[0178] Generally, the amounts of non-base agents will be sufficient
to provide final formulations in which each non-base inactive agent
represents 0.01%-50% (w/w) of the composition. Suspending agents
may represent 1%-50% (w/w) of the final formulation. Preferably,
suspending agents will represent 1%-40% and even more preferably,
they will represent 5%-30% of the final formulation. Anti-foaming
agents may represent 0.01% to 20% (w/w) of the final formulation.
More preferably, anti-foaming agents represent 0.05% to 10% of the
final formulation and even more preferably, they represent 0.1% to
5% of the final formulation.
[0179] In some preferred embodiments, the single or multiple unit
of use kits are designed to yield, after the physical mixing of
active and inactive agents, compounded pharmaceutical formulations
comprising 1%, 5%, 10% or 20% w/w of CoQ10.
[0180] The kits of the invention will provide each and every
component required for preparing a given compounded pharmaceutical
in pre-measured quantities. The measuring of each component will be
performed using current Good Manufacturing Practices (cGMP, as
legislated by the Code of Federal Regulations or CFR), as will the
packaging and labeling of each component and the final packaging
and labeling of the kit in its entirety. In this way, the kits are
standardized and variations from batch to batch will be minimal or
non-existent and the precision and accuracy in the measurement of
individual components will be improved considerably over the
methods currently used by pharmacists. Instructions may be provided
as separate from any container, but still contained in the kit.
Alternatively, instructions may be located on a container, for
example, on an exterior surface or on an interior surface such as a
lid.
[0181] Both the active and the inactive agents of the kit are
provided in containers. Since the kit will contain at least one
active and at least one inactive agent, or at least two active
agents pre-formulated with inactive agents, the minimum number of
containers in a given kit will be two. In preferred embodiments,
the maximum number of containers in a kit will be less than or
equal to four. The containers may be formed in any size or shape
useful for the mixing or transferring of components from one
container to another. For example, each container may be in the
form of vials, bottles, squeeze bottles, jars, sealed sleeves,
envelopes or pouches, tubes or blister packages or any other
suitable form provided the container is sealed so as to prevent
premature mixing of components. As used herein, a container may
also be a compartment or a chamber within a vial, a tube, ajar, or
an envelope, or a sleeve, or a blister package or a bottle,
provided that the contents of one compartment are not able to
associate physically with the contents of another compartment prior
to their deliberate mixing by a pharmacist or physician.
[0182] The invention intends to provide within a single kit all the
necessary components, containers and stirring or mixing elements
for preparing a unit of use compounded pharmaceutical without the
need for other accessories. The kits of the invention may also
contain items such as gloves or spill pads. Individuals skilled in
the art can readily modify the choice of container to suit the
individual components housed and mixed therein.
[0183] In some embodiments of the invention, the final compounded
formulation will be provided to the patient in the container
originally housing the inactive, or base, compound. In other
embodiments, the final compounded formulation will be provided in
the container originally housing the active agent. In still other
embodiments, all the necessary components for preparing a
compounded pharmaceutical are included in one container but are
physically separated within such a container. For example, an
inactive agent may be contained in the lower part of a container,
such as a jar, and may be covered by a plastic, peel-off wrap. The
active agent may be housed in this same jar, but secured to the lid
of the jar and provided in a pouch or a sleeve. The ability to
provide all components together in the smallest packaging
arrangement may be preferable in some circumstances. Mixing
elements required in the preparation of the compounded
pharmaceutical may also be located within the same container, for
example, secured to the inside surface of the lid of the
container.
[0184] In still another embodiment of the invention, active and
inactive agents are provided in adjacent compartments of a single
housing container, and are mechanically removed from these
compartments and into a third compartment. As an example, all the
chemical components necessary to prepare a particular compounded
pharmaceutical can be present in a single tube, for example, a tube
similar to a toothpaste tube having an interior which is divided
into separate compartments. Each of these compartments in turn
house a base agent or an active agent. Either the base agent or the
active agent may be premixed with an anti-foaming agent and/or a
suspending agent, as described herein. By applying pressure on the
tube as a whole, the components are made to exit their respective
compartments. They can then be mixed either in an adjacent or a
physically separate compartment. Squeezing or pressing of the
outside surface of the tube may be all that is necessary to
retrieve the individual components housed within the tube. In yet
another embodiment, the contents of both chambers of a container
can be pumped out and into a third container. In a related
embodiment, it is also envisioned that rather than requiring the
contents of each compartment to exit and flow into a third
compartment, the components may be separated by a removable sheet
or film. Thus, upon removal of such a sheet or film, the contents
of the two compartments are in contact and may require only
agitation or end-over-end inversion to become completely mixed.
This latter embodiment would eliminate the need for a mixing
element, and potentially for an exterior package particularly if
the instructions are written on the container itself.
[0185] According to some aspects of the invention, each container
may contain one or more active agents or one or more inactive
agents. For example, in some embodiments of the invention, none of
the containers may contain both an active and an inactive agent
prior to mixing by the pharmacist or physician. However, the
invention also provides for kits in which a container may contain
an active and at least one inactive agent, such as a base agent, a
suspending agent or an anti-foaming agent.
[0186] In a preferred embodiment, the active agent is provided
premixed with an inactive agent. This applies mainly when CoQ10 is
commercially available as a solid, for example a powder, and the
pre-mixing of the powder with a suspending agent facilitates the
compounding by the pharmacist or physician. In yet other
embodiments, at least two of the inactive agents may be pre-mixed
as provided in the kits of the invention.
[0187] In some embodiments, where the active agent is added to the
base component, it may be desirable to provide the base component
in a container which is only partially full. In preferred
embodiments, the container in which the base component is situated
is less than 100% full by volume. In other embodiments, the
containers are 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%,
20% or less than 20% full by volume. In other embodiments, the
active or inactive agents comprise a volume of their respective
containers ranging from 100% to greater than 1%, and every integer
there between. In preferred embodiments, the inactive agent
occupies a volume of the second container which is less than or
equal to the volume of the second container minus the volume of the
active agent.
[0188] As used according to the invention, the active and inactive
agents are physically combined by a pharmacist to produce a
compounded pharmaceutical. The components of the kit can be
combined by gentle agitation, shaking, stirring, folding or
end-over-end inversion of the first or second container. In some
instances, the proper mixing of the active and inactive agents may
be accomplished simply by adding one to the other, followed by
sealing and agitation of the container. This is especially the case
if the components are both liquids or both semi-solids. In other
instances, it may be necessary to stir the components together with
a mixing element. Mixing elements are well known to a person of
ordinary skill in the pharmaceutical arts and may include for
example, centrifuges, a mixing rod such as a glass rod, a spoon, a
spatula or a dipstick. Where required, the mixing element is
provided in the kit. The presence of a mixing element will vary
depending on the compounded pharmaceutical formulation to be made
with the components of a kit.
[0189] The final compounded pharmaceutical may be formulated into
preparations in solid, semi-solid, liquid or gaseous forms such as
tablets, capsules, powders, granules, ointments, solutions,
suppositories, inhalants and injections, and usual ways for oral,
parenteral or surgical administration. The invention also embraces
locally administering the compounded pharmaceuticals of the
invention such as, for example, as implants. These formulations may
be intended for oral, topical, mucosal, parenteral (e.g.,
injectable), rectal or vaginal administration. In preferred
embodiments, the final compounded formulations may be
self-administered.
[0190] The kits of the invention may also contain a package which
may be compartmentalized to receive in close confinement two or
more containers of the invention. In some embodiments, the package
may be box-like, being made of a moderately rigid material such as
cardboard or reinforced paper. In other embodiments, the package
may be a bag. In still other embodiments, as described herein,
there is no external packaging and all containers may be
incorporated into one of the containers housing either an active or
an inactive agent. This latter embodiment can be accomplished by
securing containers such as pouches, sleeves or sacs, containing
either active or inactive agents, as well as any mixing elements
required for the compounding, to the interior of the lid of the
main container. An individual skilled in the art can readily modify
the package to suit the individual needs of each kit and each use.
The kits of the invention further contain instructions for the
proper use of the components found therein.
[0191] The kits of the invention are intended for use in the
treatment or prevention of a number of disorders in a variety of
subjects including humans, dogs, cats, horses, fish, pigs, cows,
sheep, deer, zoo animals and laboratory animals (e.g., mice, rats,
rabbits, monkeys, etc.). The invention intends to embrace unit of
use kits containing the above preparations.
[0192] The following examples are offered by way of illustration,
not by way of limitation. While specific examples have been
provided, the above description is illustrative and not
restrictive. Any one or more of the features of the previously
described embodiments can be combined in any manner with one or
more features of any other embodiments in the present invention.
Furthermore, many variations of the invention will become apparent
to those skilled in the art upon review of the specification. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
[0193] All publications and patent documents cited in this
application are incorporated by reference in pertinent part for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted. By their citation of
various references in this document, Applicants do not admit any
particular reference is "prior art" to their invention.
EXAMPLES
[0194] The following examples serve to illustrate the invention
without limiting it thereby. It will be understood that variations
and modifications can be made without departing from the spirit and
scope of the invention.
Example 1
Materials and Methods for Apoptosis Assay
[0195] Cell lines used in the assay were SK-Me128 and nFIB. Cells
(SK-Me128 and nFIB) were seeded (5.times.10.sup.4 cells/well) into
wells containing either solely medium or medium with treatment and
placed in an incubator at 37.degree. C., 5% CO.sub.2, and under
humidified conditions for 48 hours. Each condition was performed in
duplicate and was subjected to the following protocol:
Apoptosis Analysis as Per Protocol of BD Pharmingen Annexin-VPE
Protocol
[0196] Reagents include Annexin V-PE (BD Pharmingen, San Diego,
Calif.), 7-AAD (BD Pharmingen, San Diego, Calif.), binding buffer
(10.times.: 0.1 M Hepes/NaOH, 1.4 M NaCl, 25 mM CaCl.sub.2)
[diluted to 1.times. (9 mL PBS and 1 mL binding buffer) for use in
experiment] (BD Pharmingen, San Diego, Calif.), Trypsin-EDTA
(Gibco, Grand Island, N.Y.), and desired Media.
[0197] Add 0.5 mL trypsin to each well, remove trypsin after
approximately 10 seconds, and add 0.5 mL trypsin to each well.
Place wells in an incubator, observe level of detachment under
microscope after 4 minutes, and gently tap sides and bottom to aid
in detachment. When cells detach, neutralize with 0.5 mL
serum-supplemented medium. Transfer cell solution to centrifuge
tubes, centrifuge cells at 2000 RPM for 5 minutes, aspirate
supernatant, resuspend in 6 mL PBS, and split 6 mL into three
centrifuge tubes (2 mL each). Centrifuge cells at 2000 RPM for 5
minutes, aspirate supernatant, resuspend in 1004, binding buffer
mix, add 50 .mu.L of Annexin V-PE and 50 .mu.L of 7-AAD in each
centrifuge tube, and vortex and place in the dark for 15 minutes.
Add 350 .mu.L binding buffer to each tube and perform analysis
using the flow cytometer.
[0198] A baseline was also created using freshly cultured cells
from a flask. The cells were subcultured and washed twice with cold
PBS. Subsequently, they were resuspended in 1.times. binding buffer
to a concentration of 1.times.10.sup.6 cells/mL. 100 .mu.L of cell
suspension were transferred into three test tubes for a total of
1.times.10.sup.5 per tube. One tube served as a negative control
with no staining introduced. Another was stained with only Annexin
V-PE while the final was stained with only 7-AAD. 504 of staining
solution was placed into each of the tubes. These tubes were then
placed in the dark for 15 minutes after which time, 350 .mu.L of
binding buffer were added to each. They were then subjected to
analysis by flow cytometry prior to the treated and control
cells.
Experiment 1: The Effect of Coenzyme Q10 on the Level of Apoptosis
in Human Breast Cancer Cells
TABLE-US-00002 [0199] MCF-7 MCF-7 MCF-7 Control Control Control 100
.mu.M 100 .mu.M 100 .mu.M CoQ10 CoQ10 CoQ10
[0200] Seeded 50,000 cells/well [0201] Compare to Baseline of
100,000 cells/sample in Apoptosis Assay (Annexin PI) after 72
hrs
Experiment 2: The Effect of 2-Propanol Vehicle on the Level of
Apoptosis in Melanoma Cells
TABLE-US-00003 [0202] SK-MEL 28 SK-MEL 28 SK-MEL 28 Control Control
Control Equivalent Vol. if Equivalent Vol. if Equivalent Vol. if 50
.mu.M of CoQ10 50 .mu.M of CoQ10 50 .mu.M of CoQ10 (1% 2-Propanol)
(1% 2-Propanol) (1% 2-Propanol)
[0203] Seeded 50,000 cells/well [0204] Compare to Baseline of
100,000 cells/sample in Apoptosis Assay (Annexin PI) after 48
hrs
Experiment 3: The Effect of 2-Propanol Vehicle on the Level of
Apoptosis in Neonatal Fibroblasts
TABLE-US-00004 [0205] nFIB (P) 6 nFIB (P) 6 nFIB (P) 6 Control
Control Control Equivalent Vol. if Equivalent Vol. if Equivalent
Vol. if 50 .mu.M of CoQ10 50 .mu.M of CoQ10 50 .mu.M of CoQ10 (1%
2-Propanol) (1% 2-Propanol) (1% 2-Propanol)
[0206] Seeded 50,000 cells/well [0207] Compare to Baseline of
100,000 cells/sample in Apoptosis Assay (Annexin PI) after 48
hrs
Preparation of DMEM/F12 Medium
Materials:
[0207] [0208] DMEM/F12 medium (Cat#11330-032 Gibco-Invitrogen Corp,
Grand Island, N.Y.) [0209] Siliconized Sterile Pipette tips--1 mL
and 25 mL to be used with PipettMan [0210] FBS (Fetal Bovine Serum)
Supplement (Gibco-Invitrogen Corp, Grand Island, N.Y.) [0211] PSA
(Penicillin Streptomycin Amphoterocin B)--Antimicrobrial Agent
Supplement (Cascade Biologics, Inc., Portland, Oreg.)
Procedures:
[0212] Transfer appropriate amount of FBS into DMEM/F12 (e.g., 50
mL FBS in 500 mL medium for 10% serum concentration). Add
appropriate amount of PSA to obtain a solution with a final
concentration of 100 U/mL Penicillin G, 100 .mu.g/mL streptomycin
sulfate, and 0.25 .mu.g/mL Amphotericin B (e.g., 1 mL of
500.times.PSA in 500 mL medium). Mix by pipetting and inverting
bottle. Store at 4.degree. C. until use.
Preparation of EpiLife Medium
Materials:
[0213] Siliconized Sterile Pipette tips--5 mL, 10 mL to be used
with PipettMan [0214] EpiLife Media (M-EPI-500, Cascade
Biologicals) [0215] PSA (500.times. Penicillin Streptomycin
Amphoterocin B)--Antimicrobrial Agent Supplement (R-004-10 Cascade
Biologics) [0216] EDGS (Epidermal Growth Supplement) (S-012-5
Cascade Biologics)
Procedures:
[0217] Transfer one vial of EDGS (5 mL) and PSA (1 mL) into EpiLife
Medium resulting in 100 U/mL Penicillin G, 100 .mu.g/mL
streptomycin sulfate, and 0.25 .mu.g/mL Amphotericin B (e.g. 1 mL
of 500.times.PSA in 500 mL medium). Mix by pipetting and inverting.
Store in 4.degree. C. until use.
Creating a Homogenous Solution of Q10 in Media Protocol
Materials:
[0218] Polystyrene Sterile Pipette tips--200-1000 .mu.M to be used
with automatic pipettes [0219] Siliconized Sterile Pipette tips--10
mL to be used with PipettMan [0220] 15 mL Centrifuge Tubes [0221]
Media [0222] Coenzyme Q10 (Compound Solutions, Inc., Escondido,
Calif.) [0223] 2-propanol (Cat#9083-3, J. T. Baker Chemical Co.,
Phillipsbury, N.J.)
Procedures:
[0224] Retrieve Q10 stock from -20.degree. C. storage and weigh out
approximately 4.4 mg. Transfer Q10 into a 25 mL centrifuge tube.
Add 1 mL 2-propanol to centrifuge tube. Vortex and dip in hot water
bath (55.degree. C.) to promote dissolution. Add 9 mL of media to
centrifuge tube. Vortex and dip in hot water bath (55.degree. C.)
if necessary to create a homogenous solution. This results in a 500
.mu.M Q10 solution. Make serial dilutions to treatment
concentrations.
Defrosting Cells Protocol
Materials:
[0225] Siliconized Sterile Pipette tips--1 mL, 10 mL to be used
with PipettMan [0226] 75 cm.sup.2 Cell Culture Flasks [0227] 15 mL
Centrifuge Tubes
Procedures:
[0228] Acclimate reagents to 37.degree. C. in water bath. Remove
cells from liquid nitrogen tank. Keep vial clasped in palm to
initiate defrost. Submerge in water bath at 37.degree. C. until
completely melted. Transfer cells to a 15 mL centrifuge tube with
10 mL of growth medium. Mix by pipetting. Centrifuge at 2500 RPM
for 8 minutes. Aspirate supernatant. Resuspend pellet with
appropriate medium. Mix by vortexing and pipetting to homogenize
cell suspension. Transfer to 75 cm.sup.2 Cell Culture flask(s).
Subculturing Cells Protocol
Materials:
[0229] Siliconized Sterile Pipette tips--5 mL, 10 mL to be used
with PipetteMan [0230] 75 cm2 (T75) Cell Culture Flasks [0231] 6
Well Tissue Culture Plates [0232] 15 mL Centrifuge Tubes [0233]
Media [0234] 0.05% Trypsin (Cat#25-052-C1--1.times. Trypsin-EDTA,
Cellgro by Mediatech, Herndon, Va.)
Procedures:
[0235] Acclimate reagents to 37.degree. C. in water bath. Remove
medium from cell culture flasks (cells are ready for subculture
when approximately 85% confluent). Prime by adding 1-2 mL of
trypsin to flask for 30 seconds. Remove trypsin from flask. Add 5
mL of trypsin to flask. Place flask in the incubator at 37.degree.
C. for approximately 4 minutes. Remove and observe degree of
detachment with microscope. If needed, gently tap flask to aid in
detachment. Add 5 mL of serum-containing medium. Mix by pipetting
and washing flask with cell suspension. Transfer cell suspension to
a 15 mL centrifuge tube. Vortex centrifuge tube. Centrifuge at 2500
RPM for 8 minutes. Aspirate supernatant. Resuspend pellet in
appropriate medium. Create a homogenous cell suspension by
pipetting and vortexing. Seed cells in new T75 flasks or into
wellplates for experimentation.
Counting Cells Protocol
Materials:
[0236] Beckman Coulter.RTM. Z1 Cell and Particle Counter (Beckman
Coulter, Inc., Fullerton, Calif.) [0237] Coulter Counter Vials
(Beckman Coulter, Inc.) [0238] Isoton II Diluent (#8546719, Beckman
Coulter) [0239] Coulter CLENZ (#8546929, Beckman Coulter) [0240]
Polystyrene Sterile Pipette tips--20-200 .mu.M, 200-1000 .mu.M to
be used with automatic pipettes
Procedures:
[0241] After subculture (per subculturing cells protocol described
above), pipet the desired volume suspension of cells to count
(0.25-1 mL) into Coulter Counter Vial (Beckman, Inc.) using an
automatic pipette. Insure that the Beckman Coulter.RTM. Z1 Cell and
Particle Counter is clean by using Coulter CLENZ (Beckman, Inc.,
Fullerton, Calif.) to flush. Flush apparatus once with Isoton II
Diluent. Add Isoton II Diluent to vial containing cells for a total
volume of 10 mL. Use output mode of apparatus to count cells twice
to ensure accuracy. Average counts together and calculate total
cell number per volume.
Performing in vitro Experiments Protocol
Materials:
[0242] Polystyrene Sterile Pipette tips--20-200 .mu.M, 200-1000
.mu.M to be used with automatic pipettes [0243] Siliconized Sterile
Pipette tips--5 mL, 10 mL to be used with PipettMan [0244] 75 cm2
Cell Culture Flasks [0245] 6 Well Tissue Culture Plates [0246] 15
mL Centrifuge Tubes [0247] Coulter Counter Vials (Beckman Coulter,
Inc.) [0248] 0.05% Trypsin (Cat#25-052-C1--1.times. Trypsin-EDTA,
Cellgro)
Procedures:
[0249] Acclimate reagents to 37.degree. C. in water bath. Make
stock solution of Q10 as per protocol described above for creating
a homogenous solution of Q10 in media. Perform serial dilutions to
desired concentrations. Place 2 mL media into respective wells.
Subculture flasks as per protocol described above for subculturing
cells. Resuspend cells with just enough medium to create a
homogenous cell suspension (approximately 5 mL). Determine cell
concentration as per protocol described above for counting cells.
Dilute cell suspension so that the desired amount of cells to seed
is contained within 50-100 .mu.L. Seed desired amount of cells into
each well. Incubate at 37.degree. C., 5% CO.sub.2, and under
humidified conditions for desired duration. Aspirate media from
wells. Place 0.5 mL trypsin into each well. Incubate for
approximately 4 minutes. Check for degree of detachment under
microscope. Swirl, gently tap sides, and gently knock bottom to aid
in detachment if necessary. Neutralize trypsin with 0.5 mL medium.
Pipette to aid in cell detachment and breaking of clumps. Remove
0.5 mL cell suspension and place in coulter counter vials (Beckman
Coulter, Inc.). Count cells as per protocol described above for
counting cells.
Inoculation of Animals Protocol
Materials:
[0250] Phospate buffer solution (PBS) (Gibco-Invitrogen Corp, Grand
Island, N.Y.) [0251] Polystyrene Sterile Pipette tips--20-200
.mu.M, 200-1000 .mu.M to be used with automatic pipettes [0252]
Siliconized Sterile Pipette tips--5 mL, 10 mL to be used with
PipettMan [0253] 75 cm.sup.2 Cell Culture Flasks [0254] 15 mL
Centrifuge Tubes [0255] Coulter Counter Vials (Beckman Coulter
Inc.) [0256] 0.05% trypsin (Cat#25-052-C1--1.times. Trypsin-EDTA,
Cellgro) [0257] Centrifuge tubes (2 mL) [0258] Anesthetic
(Aventin)
Procedures:
[0259] Subculture flasks as per the cell subculturing protocol
described above. After aspirating supernatant, combine pellets from
each flask diluted slightly with PBS with a 5 mL pipette. Dilute
final cell suspension to contain approximately ten million cells
per 100 .mu.L. Transfer cell suspension to micro-centrifuge tubes
(2 mL). Place in ice immediately and leave in ice until injected.
Anesthetize mice via an intraperitoneal injection with 0.3 cc
Aventin. Inoculate each animal subcutaneously with 0.1 cc cell
suspension per site. Transfer any remaining cells into a 15 mL
centrifuge tube. Dilute to 10 mL with medium. Centrifuge at 2500
RPM for 8 minutes. Aspirate supernatant. Add 10 mL media to
centrifuge tube. Create a homogenous cell suspension by pipetting
and vortexing. Seed cells in a T75 flask to ensure experimental
cell viability.
Example 2
Effect of a Topical Formulation of Coenzyme Q10 on SK-MEL28 Tumors
in Mice
[0260] Melanoma tumors were induced in mice by SK-MEL28 injection
into the subcutaneous layer. The animal study consisted of both a
control and treatment group each containing four mice. The mice
were inoculated with two tumors and the graph of FIG. 14 represents
the resulting mean mass for the tumors in each mouse. A topical
formulation of Coenzyme Q10 (10%) was applied to the tumors in the
treatment group daily for a period of 30 days. After which, the
tumors were excised and the mass was determined. The difference in
the overall mean mass of the treatment group was significant
compared to the control (P<0.05).
Example 3
Preparation of Topical CoQ10 Cream
[0261] Reagents: [0262] Phospholipon 90G (American Lechitin,
Stanford, Conn.) [0263] Glycerol [0264] BHT [0265] Ethanol [0266]
MCT [0267] lavender (Sigma-Aldrich) [0268] CoQ10 (Pure
Prescriptions, San Diego, Calif.)
Procedure:
[0269] 6 g of Phospholipon 90G (American Lechitin, Stanford, Conn.)
was dissolved in a mixture of 5.8 g of Glycerol (Sigma-Aldrich, St.
Louis, Mo.), 0.2 g BHT (Sigma-Aldrich), 4 ml ethanol
(Sigma-Aldrich), and 18 g MCT (Sigma-Aldrich) at 60.degree. C. 20 g
of CoQ10 (Pure Prescriptions) were dissolved into the resulting
mixture. 90 ml of 1 mM phosphate buffer (pH 8.2) prepared with
nitrogen saturated water and 0.2 ml of lavender (Sigma-Aldrich)
were added and the mixture was blended in a high speed blender at
12,000 RPM to form a cream. The cream was stored at 4.degree. C.
until used.
Example 4
Apoptosis Analysis for JC-1 Stain
[0270] Apoptosis was measured using a mitochondrial membrane dye
JC-1,5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanin-
e chloride (Molecular Probe, Eugene, Oreg.). Treatments consisting
of DMEM-F12 media supplemented with 1.times.PSA, 5% FBS and 0, 50,
100, and 200 .mu.M concentrations of coenzyme Q10 were prepared in
60.times.15 mm tissue culture dishes (Costar--Cambride, Mass.).
PC-3 cells were seeded at 500,000 cells per dish and incubated for
24 hours. The cells were trypsinized using 2 mL trypsin-EDTA and
subjected to centrifugation at 2,500 rpm for 8 minutes. They were
resuspended in 1 mL of Ham's F12 medium lacking serum and phenol
red (Cascade Biologics, Inc-Portland, Oreg.) and promptly placed on
ice. A 1 mg/ml stock solution of JC-1 was prepared using sterile
DMSO and 10 .mu.L, was added to each cell suspension while gently
vortexing. The cells were incubated at 37.degree. C. for 15 min,
diluted with 4 ml of Ham's F12 medium and centrifuged at 600 rpm
for 7 min. Resuspended in 5 ml of cold PBS (Gibco-Grand Island,
N.Y.), the cells were centrifuged again at 600 rpm for 7 min. The
cell pellet was then suspended in 1 ml of cold PBS and transferred
to nylon filter top flow cytometry tubes covered with foil to
prevent light penetration. The samples were analyzed by flow
cytometry for changes in uptake of fluorescent dye. The monomer
JC-1 displays green fluorescence (.lamda..sub.em=527 nm) while the
J-aggregates display red fluorescence (.lamda..sub.em=590 nm).
Permeabilized mitochondria accumulate the JC-1 monomer dye prior to
and during apoptosis.
OTHER EMBODIMENTS
[0271] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspect, advantages, and modifications are
within the scope of the following claims.
* * * * *