U.S. patent application number 15/184876 was filed with the patent office on 2016-10-06 for melanoma chemoprevention.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Jan H. Beumer, Charles K. Brown, John M. Kirkwood, Shivendra V. Singh.
Application Number | 20160287548 15/184876 |
Document ID | / |
Family ID | 49514916 |
Filed Date | 2016-10-06 |
United States Patent
Application |
20160287548 |
Kind Code |
A1 |
Beumer; Jan H. ; et
al. |
October 6, 2016 |
MELANOMA CHEMOPREVENTION
Abstract
Disclosed herein are methods and uses for preventing melanoma,
reducing progression of atypical nevi, and inducing cell cycle
arrest and/or apoptosis in a melanoma cell through oral and enteral
administration of sulforaphane to subjects indicated to be at risk
due to factor(s) such as medical history of atypical nevi,
melanoma, or UV exposure. Sulforaphane can be administered orally
as a safe and well-tolerated natural agent as a chemopreventive
strategy in individuals with atypical melanocytic nevi.
Inventors: |
Beumer; Jan H.; (Aspinwall,
PA) ; Kirkwood; John M.; (Gibsonia, PA) ;
Singh; Shivendra V.; (Wexford, PA) ; Brown; Charles
K.; (Genoa City, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education |
Pittsburgh |
PA |
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Pittsburgh
PA
|
Family ID: |
49514916 |
Appl. No.: |
15/184876 |
Filed: |
June 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14398713 |
Nov 3, 2014 |
9393225 |
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PCT/US2013/039518 |
May 3, 2013 |
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15184876 |
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61643098 |
May 4, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/52 20130101;
A61P 35/04 20180101; A61P 17/00 20180101; G01N 33/6893 20130101;
G01N 2333/4706 20130101; A61K 9/0053 20130101; A61K 31/26
20130101 |
International
Class: |
A61K 31/26 20060101
A61K031/26; G01N 33/68 20060101 G01N033/68; A61K 9/00 20060101
A61K009/00 |
Claims
1. A method of preventing progression of atypical nevi in a
subject, comprising administering orally to a subject having
atypical nevi a therapeutically effective amount of a
pharmaceutical composition comprising sulforaphane, thereby
reducing progression of the atypical nevi.
2. A method of preventing melanoma in a subject, comprising
administering orally to a subject a therapeutically effective
amount of a pharmaceutical composition comprising sulforaphane,
thereby preventing melanoma in the subject.
3. The method of claim 1, wherein preventing progression comprises
preventing emergence of additional atypical nevi in the
subject.
4. The method of claim 1, wherein preventing progression comprises
preventing transition of atypical nevi from morphologically less
atypical to morphologically more atypical on a tiered scale.
5. The method of claim 1, comprising administering the sulforaphane
as a tea or as a gel capsule.
6. The method of claim 1, comprising administering about 50 to
about 400 .mu.M of sulforaphane.
7. The method of claim 1, comprising administering about 0.5 to
about 10 .mu.M of sulforaphane per kilogram of bodyweight of the
subject.
8. The method of claim 1, comprising administering about 50, about
100, or about 200 .mu.M of sulforaphane.
9. The method of claim 1, comprising administering about 0.5, about
2.0, or about 4.0 .mu.M of sulforaphane per kilogram of bodyweight
of the subject.
10. The method of claim 1, comprising administering the
sulforaphane in repeated doses.
11. The method of claim 1, comprising administering the
sulforaphane thrice daily, twice daily, daily, bi-weekly, or
weekly.
12. The method of claim 1, wherein administering is repeated over
about 10 days to about 6 months.
13. The method of claim 2, wherein the subject has a family history
of melanoma or dysplastic nevi syndrome.
14. The method of claim 1, wherein the subject has two or more
atypical nevi.
15. The method of claim 1, wherein the subject has two or more
atypical nevi that are greater than 4 millimeters in diameter.
16. The method of claim 1, wherein the subject has two or more
atypical nevi, and wherein one or more of the nevi have been
morphologically classified on a tiered scale.
17. The method of claim 2, wherein the subject has a loss of
function mutation in an MC1R gene.
18. The method of claim 1, further comprising measuring STAT1 or
STAT3 expression in a sample from the subject.
19. The method of claim 18, wherein the sample is a biopsy.
20. The method of claim 19, wherein measuring STAT1 or STAT3
expression comprises measuring with an immunoassay.
21. The method of claim 1, wherein the subject has a loss of
function mutation in an MC1R gene.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
14/398,713, filed on Nov. 3, 2014, which is a .sctn.371 U.S.
national stage of PCT/US2013/039518, filed May 3, 2013, which was
published in English under PCT Article 2(2), and claims the benefit
of U.S. Provisional Application No. 61/643,098, filed May 4, 2012.
The prior applications are incorporated by reference herein.
FIELD
[0002] This disclosure is related to the use of sulforaphane
administered orally for the prevention of melanoma, such as in
specific populations of subjects that are at risk for melanoma.
BACKGROUND
[0003] Safe and effective chemoprevention agents have enormous
potential to improve human health. This was first demonstrated
convincingly in cardiovascular disease, where modifiable risk
factors such as high blood pressure and elevated cholesterol levels
were identified and targeted for pharmacological management. The
result has been the reduction of both the morbidity and mortality
associated with heart disease. For some cancers, the promise of
chemoprevention has already been realized. Recurrence of breast
cancer is being reduced by treatment with tamoxifen, and the
incidence of colon cancer is being reduced significantly with
NSAIDs.
[0004] Melanoma is less common than other skin cancers, but it is
much more dangerous if it is not detected early. Melanoma causes
the majority (75%) of deaths related to skin cancer. Worldwide,
doctors diagnose about 160,000 new cases of melanoma yearly. The
diagnosis is more frequent in women than in men. In women, the most
common site is the legs and melanomas in men are most common on the
back Melanomas are aggressive and frequently metastatic tumors
develop. Melanocytic nevi can be broadly categorized into normal
nevocellular nevi (or benign neoplasms of melanocytes) and atypical
nevi, which are also known as dysplastic nevi.
[0005] Minimizing exposure to sources of ultraviolet radiation,
such as from the sun and sunbeds, following sun protection
measures, and wearing sun protective clothing can offer protection
from melanoma. However, a need remains for further preventive
measures, such as to prevent melanoma in subjects that are
pre-disposed to develop this disease.
SUMMARY
[0006] It is disclosed herein that pharmaceutical compositions
including sulforaphane (SFN) can be administered to a subject to
reduce progression of atypical nevi and/or to prevent melanoma. The
subjects can have atypical nevi, can have a dysplastic nevus
syndrome, or can be at risk for developing melanoma, such as due to
a family history.
[0007] In some embodiments, methods or uses are provided for
reducing progression of atypical nevi in a subject. In an example
embodiment, a therapeutically effective amount of a pharmaceutical
composition including SFN is administered orally to a subject
having atypical nevi, thereby reducing progression of the atypical
nevi in the subject.
[0008] In additional embodiments, methods or uses are provided for
preventing melanoma in a subject. In an example embodiment, a
therapeutically effective amount of a pharmaceutical composition
including SFN is administered orally to a subject having atypical
nevi, or otherwise at risk for developing melanoma, thereby
preventing melanoma from developing in the subject.
[0009] In additional embodiments, methods or uses are provided to
induce cell cycle arrest and/or apoptosis of a dysplastic skin cell
and/or melanoma cell. In an example embodiment, a pharmaceutical
composition including SFN is administered orally to a subject
having a melanoma cell, thereby inducing cell cycle arrest and/or
apoptosis of the melanoma cell of the subject.
[0010] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 is a set of bar graphs showing that SFN inhibits
melanoma cell growth in vitro.
[0012] FIG. 2 is a set of digital images demonstrating that SFN
inhibits melanoma cell Cyclin B1, Cdk1, and Cdc25 in vitro.
[0013] FIG. 3 is a set of digital images and a bar graph
demonstrating that oral SFN modulates STAT3 in human to mice
melanoma xenotransplants.
[0014] FIGS. 4A-4C are a bar graph (A), plot (B) and table (C)
demonstrating the effect of SFN treatment on proliferation of
SK-MEL 31 cells. Proliferation was measured by (A) trypan blue dye
exclusion assay and (B) FACS cell cycle distribution assay after
PI. For trypan blue exclusion assay, SK-MEL 31 cells were exposed
to 10, 20 or 40 .mu.M SFN or DMSO (control) for 24, 48 and 72
hours. Data are presented as mean.+-.SE (n=3) from a representative
experiment. For cell cycle distribution assay, cells were exposed
to 20 .mu.M SFN or DMSO (control) for 24 hours, results were
presented as mean.+-.SE (n=3) from a representative experiment. All
experiments were repeated at least twice with comparable results.
The notation (*) indicates significantly different result compared
with control (p<0.01) by one way ANOVA, while the notation (#)
indicates a significantly different compared with control
(p<0.01) by Student's t-test.
[0015] FIGS. 5A-5C are a set of bar graphs (A and C) and a digital
image (B) demonstrating the effect of SFN treatment on apoptosis
induction. (A) Quantification of cytoplasmic histone associated DNA
fragments following a 24 h exposure to DMSO (control) or SFN (2.5,
5 or 10 .mu.M), data are mean.+-.SE of three determinations from a
representative experiment. (B) Western blot analysis for cleaved
PARP (89 kDa) using lysates from SK-MEL 31 cells exposed to 20 mM
SFN after different time intervals (each lane loaded with 60 .mu.g
of lysate protein). (C) Condensed nucleus is the typical morphology
change of apoptosis, the condensed nucleus ratio increased
significantly after SFN treatment. The notation (#) indicates a
significantly different compared with control, p<0.05; and the
notation (*) indicates a significantly different compared with
control, p<0.01. All experiments were repeated twice and the
results were comparable.
[0016] FIGS. 6A-6C are a plot (A) and a set of bar graphs (B and C)
demonstrating the effect of oral administration of SFN on growth of
SK-MEL 31 tumor xenografts in female nude mice. The results
achieved with regard to (A) tumor volume, (B) tumor wet weight, and
(C) apoptotic bodies by TUNEL assay are shown. Administration of
SFN decreased the tumor burden accompanied by induced apoptosis.
Data are presented as mean.+-.SE. The notation (*) indicates a
significantly different compared with control, p<0.01 by
Student's t-test, while the notation (#) indicates a significantly
different result compared with control, p<0.05 by Student's
t-test.
[0017] FIGS. 7A-7B are a set of digital images demonstrating
expression of Bcl-2 family using lysates from SK-MEL 31 cells
exposed to 20 .mu.M SFN for specified time intervals; and
expression of IAPs and Smac, cytochrome c released from
mitochondria after SFN treatment. FIG. 7A shows representative
western blots for expression of Bcl-2 family using lysates from
SK-MEL 31 cells exposed to 20 .mu.M SFN for specified time
intervals. Equal amounts of lysate protein (10 .mu.g for Bax and 40
.mu.g for other molecules) were subjected to gel electrophoresis.
FIG. 7B shows Western blot assays of IAPs and Smac, cytochrome c
released from mitochondria after SFN treatment. Whole cell lysate
were used for cIAP1, XIAP (40 .mu.g for each lane), while cytosolic
fraction were prepared for Smac and cytochrome c assay (equal
volume of lysate for each lane), the target protein level changes
were corrected with corresponding actin level for each lane. All
experiments were repeated twice, with comparable results.
[0018] FIGS. 8A-8E are a set of bar graphs (A, B and D) and a set
of digital images (C and E) demonstrating involvement of caspases
in SFN induced apoptosis in SK-MEL 31. (A) and (B): Colormetric
assays of caspase-8 and caspase-9 activity after SFN treatment for
16 and 24 hours; the caspase activity was presented as the
percentage of control group. (C) Western blot analysis for cleaved
caspase-3, caspase-8 and caspase-9 using lysates from SK-MEL 31
cells exposed to 20 mM SFN for specified time intervals (equal
amounts (60 .mu.g) of lysate protein subjected to gel
electrophoresis). (D) ELISA detection of cytoplasmic histone
associated DNA fragments level after introduction of caspase
inhibitors before SFN treatment. (E) Western blot assay of
caspase-3 cleavage after introduction of caspase inhibitors before
SFN treatment. All experiments were repeated twice and results were
comparable. Data are mean.+-.SE of three determinations in (A),
(B), (D) from a representative experiment. The notation (*)
indicates a significantly different compared with control (caspase
activity assay) or SFN treated group (cell Death ELISA) (p<0.01)
by one-way ANOVA.
[0019] FIGS. 9A-9E are a set of digital images (A, C, D and E) and
a bar graph (B) demonstrating that Bcl-XL overexpression protects
against SFN induced cell apoptosis. (A) shows immunoblotting for
Bcl-XL using lysates from S-KMEL31 cells transiently transfected
with pSFFV-Bcl-XL and pSFFV-neo plasmids. (B) shows ELISA detection
of cytoplasmic histone associated DNA fragments level in SK-MEL31,
SK-MEL31/neo and SK-MEL 31/Bcl-XL cells following exposure to SFN
10 .mu.M for 24 hours. Data are mean.+-.SE, n=3. The results are
significantly different compared with control, with the notation
(*) signifying significance at p<0.01 and the notation (#)
indicating significance at p<0.05 by one-way ANOVA. (C) shows
cleavage of PARP after exposure to SFN 20 .mu.M for 24 hours. (D)
shows cleavage of pro-caspase 3 and 9 after exposure to SFN 20
.mu.M for 24 hours. (E) shows release of cytochrome-c and Smac from
mitochondria after exposure to SFN 20 .mu.M for 24 hours.
[0020] FIG. 10 is a flowchart demonstrating a proposed mechanism
for SFN induced apoptosis in in SK-MEL 31 cells based on the
results. SFN induced apoptosis in SK-MEL 31 cells was associated
with a downregulation of anti-apoptotic Bcl-2 family members
(Bcl-XL), IAPs (XIAP, cIAP1). Activation of caspase-8, -9, -3, and
cleavage of PARP were also involved in this process. Release of
cytochrome c and Smac into the cytoplasm suggests the role of
mitochondria in apoptotic signaling.
[0021] FIG. 11 is a schematic diagram of an exemplary protocol for
the administration of SFN to a subject with atypical nevi.
[0022] FIG. 12 is a table showing scheduled evaluations for
subjects evaluated by the protocol presented in FIG. 11.
DESCRIPTION OF EMBODIMENTS
[0023] There is a genetic predisposition to melanoma that requires
unique preventive measures. The presence of atypical nevi is
positively correlated with increased risk of melanoma and occurs in
two main settings. Subjects with familial melanoma or dysplastic
nevus syndrome exhibit an autosomal dominant trait with a lifetime
melanoma risk approaching 100%. Limiting UV radiation exposure
cannot serve as the sole agent of prevention in these
individuals.
[0024] In light of excellent survival rates for melanoma discovered
while still in situ compared to the poorer prognosis associated
with more advanced disease, efforts are warranted to identify means
of primary prevention of melanoma. Given the continually rising
incidence of melanoma, the ineffectiveness of UV radiation
protective strategies and prevalence of genetic pre-disposing
factors, chemoprevention serves as an attractive strategy for
primary prevention of melanoma.
[0025] Methods are provided herein for reducing the progression of
nevi. Methods are also disclosed for preventing melanoma, including
reducing the appearance of melanoma precursor lesions. These
methods include the oral administration of Sulforaphane (SFN).
Sulforaphane (SFN, 1-isothiocyanato-4-(methylsulfinyl)-butane;
CH3--SO--(CH2)4--N.dbd.C.dbd.S), a constituent of cruciferous
vegetables including broccoli is generated through catalytic
hydrolysis of glucoraphanin by myrosinase, an endogenous enzyme and
released upon damage of the plant cells during digestive processing
(cutting or chewing). See Zhang et al., Proc. Natl Acad. Sci. USA
(1992) 89: 2399-2403. SFN affects biomarkers in the progression
from atypical nevi to melanoma. One biomarker is the effect of SFN
on the Signal Transducer and Activation of Transcription (STAT)
pathways whose dysregulation promotes tumorigenesis in melanoma and
other solid tumors. SFN inhibits carcinogenesis through several
different mechanisms within the initiation, promotion, and
progression phases of cancer development in multiple types of
tumors.
[0026] SFN can be administered orally as a safe and well-tolerated
agent for chemoprevention in individuals with atypical melanocytic
nevi which are associated with an increased predisposition towards
developing melanoma.
Abbreviations
[0027] To facilitate review of the various embodiments of this
disclosure, the following explanations of abbreviations are
provided:
[0028] BSE Broccoli Sprout Extract
[0029] C Celsius
[0030] CI Confidence interval
[0031] ELISA Enzyme-linked immunosorbent assay
[0032] FAMM Familial atypical multiple mole-melanoma
[0033] h hour(s)
[0034] MC1R MelanoCortin 1 Receptor
[0035] .mu.M microMole
[0036] PD Pharmacodynamics
[0037] PK Pharmacokinetics
[0038] RIA Radioimmunoassay
[0039] SAE rate Small Area Estimate rate
[0040] SE Standard Error
[0041] SFN Sulforaphane
[0042] STAT Signal Transducer and Activation of Transcription
[0043] TUNEL Terminal deoxynucleotidyl transferase dUTP Nick End
Labeling
Terms
[0044] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0045] Administering: Introducing a pharmaceutical composition to a
subject. In one example, a pharmaceutical composition is
administered orally. Examples of oral administration include
swallowing a liquid, a gel capsule or other pill or other
solid.
[0046] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects.
[0047] Biopsy: A sample collection technique in which tissue is
removed from a subject and sent to a pathologist to render a
diagnosis. As an example, in a skin biopsy, a portion of the skin
of a subject is removed. As an example, a skin biopsy may be
performed under local anesthetic. In other examples, a skin biopsy
may be a shave biopsy, a punch biopsy, an excisional biopsy, or an
incisional biopsy, among others. The vast majority of nevi and
melanoma precursor lesions are amenable to visual/photographic
identification/documentation and may be biopsied for
histopathologic evaluation, as well as for the performance of
molecular biologic and immunohistologic testing for candidate
markers of progression.
[0048] Buccal administration: Administration that involves
absorption of a drug through membranes in the mouth.
[0049] Chemoprevention: Prophylactic or preventive use of natural
or synthetic agents to delay, reverse, suppress or prevent
premalignant molecular or histological lesions from progressing to
invasive cancer. The concept applies to primary prevention in
high-risk individuals, secondary prevention in those with
premalignant conditions, and tertiary prevention in aiding those
with a cured malignancy from developing a second primary
cancer.
[0050] A chemopreventive agent is any biological or chemical agent
with prophylactic or preventive usefulness in the treatment of
diseases characterized by abnormal cell growth. Such diseases
include tumors, neoplasms, and cancer as well as diseases
characterized by hyperplastic growth such as psoriasis. Combination
chemoprevention is the prophylactic or preventive administration of
more than one agent to delay, reverse, suppress or prevent
premalignant molecular or histological lesions from progressing to
invasive cancer. One example is the administration of SFN for the
prevention of melanoma.
[0051] Dermatoscopy: Examination of skin lesions with a
dermatoscope. This traditionally consists of a magnifier (typically
.times.10), a non-polarized light source, a transparent plate and a
liquid medium between the instrument and the skin, and allows
inspection of skin lesions unobstructed by skin surface
reflections. Modern dermatoscopes dispense with the use of liquid
medium and instead use polarized light to cancel out skin surface
reflections. Dermatoscopy is also known as dermoscopy or
epiluminescence microscopy. For example, a dermatoscope can be
useful to dermatologists in determining the degree of atypia of a
nevus without or prior to resorting to biopsy. Dermatoscopy can be
enabled with analog and/or digital imaging (videodermatoscopy).
Digital dermatoscopy images can be stored and compared to later
images from the same subject or from other subjects. Common systems
for digital dermoscopy are Fotofinder, Molemax or Easyscan.
[0052] Diagnostic: Identifying the presence or nature of a
biological or medical condition, such as, but not limited to,
presence of a genetic mutation, systemic or localized concentration
in a subject of an administered pharmaceutical composition,
occurrence of dysplastic nevus syndrome, or occurrence of
melanoma.
[0053] Dysplastic nevus syndrome: A medical condition characterized
by the presence on the skin of multiple dysplastic, unusual, or
atypical nevi or moles. In some examples, the syndrome occurs in
multiple family members, possibly over multiple generations,
resulting in a family history of dysplastic nevus syndrome or of
melanoma. In some examples, individuals may be at increased risk of
melanoma versus the general population. "Dysplastic nevus syndrome"
is also known as "Familial atypical multiple mole-melanoma (FAMM)
syndrome" and "familial melanoma syndrome."
[0054] Enteral administration: Administration that involves
absorption of a drug through the gastrointestinal tract.
[0055] Immunoassay: A biochemical test in which antibodies are used
to target molecules to determine their presence in a sample from a
subject. Secondary marker(s) may be used to amplify and detect the
presence of primary antibody. The secondary marker may be another
antibody, agent, or reaction for detecting and amplifying the
resulting signal indicating the presence of the primary antibody.
Examples of specific types of immunoassay include ELISA, Western
blot, or RIA assay.
[0056] Isolated: An "isolated" chemical or biological component,
such as a compound, nucleic acid, protein (including antibodies),
or organelle, has been substantially separated or purified away
from other chemical or biological components in the environment
(such as an animal, a plant, or a cell) in which the component
naturally occurs. Various techniques may be used for isolating,
including excising, biopsying, extracting, purifying, and
concentrating. In an example, SFN can be extracted from certain
agricultural products (for example broccoli, brussel sprouts,
cabbage, cauliflower, collard or mustard greens, kohlrabi, bok
choy, rapini, turnips, rutabaga, arugula, certain radishes, and
watercress). It may then be purified of contaminants and potential
toxins, concentrated, and quantified as to both amount and final
concentration (or any combination of these steps).
[0057] MC1R: MelanoCortin 1 Receptor, which is the receptor of
alpha-melanocyte stimulating hormone. The "MC1R gene" encodes MC1R.
MC1R is a major genetic determinant of UV-induced melanocyte
oxidative responses, robustness to DNA damage, and melanoma risk or
susceptibility. MC1R may be essential for maintenance of a normal
redox state in melanocytes, and possibly more so in nevi that are
under constant oxidative stress. Some mutations in the MC1R gene
can result in a loss of gene or protein function and may be
associated with increased risk of melanoma. As a possible genetic
determinant of melanoma risk, MC1R may be used in some examples as
an indicator in subject selection for SFN melanoma prevention
therapy. In some examples, MC1R may be sequenced from saliva
samples, and loss of function mutations may be detected. An
exemplary nucleic acid sequence of a human MC1R gene is presented
in GENBANK.RTM. Accession No. BC007856.2 as of Jul. 15, 2006,
incorporated herein by reference.
[0058] Melanoma: A form of cancer that originates in melanocytes
(cells that make the pigment melanin). Melanocytes are found
primarily in the skin, but are also present in the bowel and eye.
Melanoma in the skin includes superficial spreading melanoma,
nodular melanoma, acral lentiginous melanoma, and lentigo maligna
(melanoma). Any of the above types may produce melanin or can be
amelanotic. Similarly, any subtype may show desmoplasia (dense
fibrous reaction with neurotropism) which is a marker of aggressive
behavior and a tendency to local recurrence. Other melanomas
include clear cell sarcoma, mucosal melanoma, and uveal
melanoma.
[0059] Features that affect prognosis are tumor thickness in
millimeters (Breslow's depth), depth related to skin structures
(Clark level), type of melanoma, presence of ulceration, presence
of lymphatic/perineural invasion, presence of tumor infiltrating
lymphocytes (if present, prognosis is better), location of lesion,
presence of satellite lesions, and presence of regional or distant
metastasis.
[0060] Nevi: Melanocytic nevi may be broadly categorized into
normal nevocellular nevi, (or benign neoplasms of melanocytes), and
atypical nevi. A neoplasm is a new growth or shape in a tissue. A
distinction may be drawn between common acquired nevi, which are
smaller than 3 mm, symmetrical, well-defined, and of uniform
tan-brown coloration, and atypical nevi which are generally greater
than 5 mm in diameter, often asymmetrical, with indistinct or
irregular borders, and of variegated color, often including pink
and red. "Atypical nevi" is the clinical description of the same
lesions known pathologically as "dysplastic nevi." Histopathology
criteria essential for diagnosis include thickness of the lesion,
extent of penetration, degree of ulceration, presence of
lymphocytic infiltrates.
[0061] A six-tiered scale (A-F) may be used to classify nevi based
on the degree of atypical morphology observed in a nevus ("F" being
the least atypical, and "A" being the most atypical). Such
morphological observations may be conducted visually or
photographically, with/without magnification, with/without applied
contrasting agents, in vivo on the skin of a subject or in situ
using a sample (e.g. biopsy) from a subject. As an example, one
purpose of the present technology is to reduce the progression of
nevi from less atypical ("F") toward more atypical ("A"). As a
further example, a tiered scale (for example A-F) may define less
to more atypical conditions as a progression with respect to one or
more of the following possible nevus properties in any combination:
asymmetry, distinctiveness and/or regularity of borders,
variegation in color, thickness, penetration, ulceration,
lymphocytic infiltration. These are just example properties, and
other properties may be useful. As a further example, this
progression can be associated with the occurrence of melanoma, so
that reducing or slowing this progression can be associated with
prevention of melanoma. For scoring and classification methods, see
Wang et al., Journal of Investigative Dermatology (2008) 128:
1997-2002, which is incorporated herein in its entirety.
[0062] Pharmaceutical composition: A chemical or biological
compound or mixture capable of inducing a desired prophylactic,
preventive, or therapeutic effect when properly administered to a
subject. Such a composition is prepared consistently with accepted
pharmaceutical practice to promote safety of the subject and
decrease risk of adverse reactions in the subject after
administration. Generally, a pharmaceutical composition includes a
pharmaceutically acceptable carrier.
[0063] In examples herein that employ pharmaceutically acceptable
carriers, these are the conventional carriers. Remington's
Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co.,
Easton, Pa., 15th Edition, 1975, describes compositions suitable in
some examples for pharmaceutical delivery of the compound herein
disclosed.
[0064] In general, the nature of a carrier will depend on the
desired final nature of the composition and the mode of
administration being employed. For solid compositions (such as
powder, pill, tablet, or capsule forms), conventional non-toxic
solid carriers can include, for example, pharmaceutical grades of
mannitol, lactose, starch, or magnesium stearate. In addition to
biologically neutral carriers, pharmaceutical compositions to be
administered can contain minor amounts of non-toxic auxiliary
substances, such as wetting or emulsifying agents, preservatives,
and pH buffering agents and the like, for example sodium acetate or
sorbitan monolaurate.
[0065] Preventing, treating or ameliorating a disease: "Preventing"
a disease refers to precluding the onset or inhibiting the full
development of a disease such as melanoma and is a form of
prophylaxis. "Treating" refers to a therapeutic intervention that
ameliorates a sign or symptom of a disease or pathological
condition such as melanoma after it has begun to develop.
"Ameliorating" refers to the reduction in the number or severity of
signs or symptoms of a disease, such as cancer.
[0066] Sample: A biopsy, serum, blood, plasma or other substance
from an animal (e.g., human) that includes biomolecules and
antibodies representative of those present in the animal. Samples
can include processed blood samples, secretions, and the like. As
an example, a sample may be tissue from a nevus harvested during a
biopsy and then subjected to one or more visual inspections and/or
biochemical assays.
[0067] STAT1 and STAT3: Signal Transducer and Activation of
Transcription (STAT) proteins are a collection of transcription
factors activated by tyrosine phosphorylation, which participate in
normal cellular events. As examples, of the 7 known STAT proteins,
STAT 1, 3, 5a and 5b have important roles in controlling cell-cycle
progression and apoptosis. For example, STAT1 is considered a tumor
suppressor gene as it inhibits growth and acts as a mediator of
apoptosis. And as another example, STAT3 has been shown to
contribute to oncogenesis by upregulating genes involved with
apoptosis inhibition, cell cycle regulation, and induction of
angiogenesis. STAT3 also participates in the regulation of tumor
immune evasion by inhibiting expression of proinflammatory
mediators while promoting expression of immune-suppressing factors.
When the STAT proteins become dysregulated, the resultant aberrant
cellular proliferation is known to contribute to carcinogenesis.
Atypia in nevi correlate with the expression of STAT1 and STAT3 in
melanocytic nevi of subjects with melanoma. In some examples, STAT
expression may be analyzed immunohistologically in biopsy tissue,
for example in biopsies of nevi. An example human STAT1 gene is
presented in GENBANK.RTM. Accession No. BC002704.2 as of Jul. 15,
2006. An example human STAT3 gene is presented in GENBANK.RTM.
Accession No. BC000627.2 as of Jul. 15, 2006.
[0068] Subject: Living multi-cellular vertebrate organisms, a
category that includes both human and veterinary subjects,
including human and non-human mammals.
[0069] Therapeutically effective amount: A quantity of a specific
substance that alone, or together with a pharmaceutically
acceptable carrier or one or more additional pharmaceutical agents,
is sufficient to achieve a desired effect in a subject being
treated. For instance, this can be the amount of SFN necessary to
prevent, reduce, inhibit, or suppress growth of an atypical nevi or
a tumor. When administered to a subject, a dosage will generally be
used that will achieve target tissue concentrations (for example,
systemically or in nevi) that have been shown to achieve a desired
effect, for example reducing the progression of nevi from less to
more atypical, or preventing melanoma.
[0070] Effective amounts of a pharmaceutical agent can be
determined in many different ways, such as by assaying for
improvement or maintenance of a physiological condition of a
subject, for example a subject having dysplastic nevus syndrome.
Effective amounts also can be determined through various in vitro,
in vivo, or in situ assays.
[0071] Therapeutic agents can be administered in a single dose, or
in several doses, for example thrice daily, twice daily, daily,
bi-weekly, weekly, monthly, or bi-monthly, during a course of
treatment. However, the effective amount can be dependent on the
source of the pharmaceutical composition, the subject being
treated, the severity and type of the condition being treated, and
the manner of administration.
[0072] Unless explained otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this disclosure belongs.
In case of conflict, the present specification, including
explanations of terms, will control. Explanation of common terms
and methods in pharmacology may be found in Remington's
Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co.,
Easton, Pa., 15th Edition, 1975 and Goodman & Gilman's The
Pharmacological Basis of Therapeutics, by L. Brunton et al., The
McGraw Hill Companies, Inc., Twelfth Edition (2011).
[0073] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety for all purposes.
[0074] Although methods and materials similar or equivalent to
those described herein can be used to practice or test the
disclosed technology, suitable methods and materials are described
below. The materials, methods, and examples are illustrative only
and not intended to be limiting.
[0075] The singular forms "a," "an," and "the" refer to one or more
than one, unless the context clearly dictates otherwise. For
example, the term "comprising a specimen" includes single or plural
specimens and is considered equivalent to the phrase "comprising at
least one specimen." The term "or" refers to a single element of
stated alternative elements or a combination of two or more
elements, unless the context clearly indicates otherwise. As used
herein, "comprises" means "includes." Thus, "comprising A or B,"
means "including A, B, or A and B," without excluding additional
elements.
[0076] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present disclosure, suitable methods and materials are
described below. All publications, patent applications, patents,
GENBANK.RTM. Accession numbers and other references mentioned
herein are incorporated by reference in their entirety. In case of
conflict, the present specification, including explanations of
terms, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
SFN and Pharmaceutical Compositions
[0077] The chemical structure of Sulforaphane (SFN) is provided
below, and Table 1 follows, listing example analogues of SFN.
##STR00001##
TABLE-US-00001 TABLE 1 Chemical formula of example SFN analogues
Common Chemical name name Chemical formula 3-(Methylthio)-propyl
Iberverin CH.sub.3--S--(CH.sub.2).sub.3--N.dbd.C.dbd.S
isothiocyanate 4-(Methylthio)-butyl Erucin
CH.sub.3--S--(CH.sub.2).sub.4--N.dbd.C.dbd.S isothiocyanate
5-(Methylthio)-pentyl Berteroin
CH.sub.3--S--(CH.sub.2).sub.5--N.dbd.C.dbd.S isothiocyanate
3-(Methylsulfinyl)-propyl Iberin
CH.sub.3--SO--(CH.sub.2).sub.3--N.dbd.C.dbd.S isothiocyanate
4-(Methylsulfinyl)-butyl Sulforaphane
CH.sub.3--SO--(CH.sub.2).sub.4--N.dbd.C.dbd.S isothiocyanate
5-(Methylsulfinyl)-pentyl Alyssin
CH.sub.3--SO--(CH.sub.2).sub.5--N.dbd.C.dbd.S isothiocyanate
3-(Methylsulfonyl)- Cheirolin
CH.sub.3--SO.sub.2--(CH.sub.2).sub.3--N.dbd.C.dbd.S propyl
isothiocyanate 4-(Methylsulfonyl)-butyl Erysolin
CH.sub.3--SO.sub.2--(CH.sub.2).sub.4--N.dbd.C.dbd.S isothiocyanate
5-(Methylsulfonyl)- Alyssin
CH.sub.3--SO.sub.2--(CH.sub.2).sub.5--N.dbd.C.dbd.S pentyl
isothiocyanate sulfone
[0078] SFN is found in cruciferous vegetables such as cabbage,
broccoli, broccoli sprouts, brussel sprouts, cauliflower,
cauliflower sprouts, bok choy, kale, collards, arugula, kohlrabi,
mustard, turnip, red radish and watercress. In the plant, it is
present in bound form as glucoraphanin, a glucosinolate.
D,L-Sulforaphane is a synthetic analogue of broccoli-derived
L-isomer. Sulforaphane is often formed from glucoraphanin on plant
cell damage via an enzymatic reaction. Various synthetic methods of
producing sulforaphane are known in the art. Sulforaphane was
synthesized as early as 1948 by Schimd and Karrer (Schimd H. and
Karrer, P.; Helvetica Chimica Acta. 1948; 31; 6: 1497-1505).
Stabilized forms of sulforaphane have also been produced, see U.S.
Published Patent Application No. 2008/0176942, Jul. 24, 2008,
incorporated herein by reference.
[0079] Additional methods are known (see U.S. Pat. No. 5,882,646
and U.S. Published Patent Application No. 2009/0247477). An
exemplary non-limiting method for producing sulforaphane includes
extracting three-day-old broccoli sprouts with hot water, treating
with myrosinase and freeze drying the treated extract under
pharmaceutically appropriate manufacturing conditions, see
Cornblatt et al., Carcinogenesis (2007) 28: 1485-1490, which is
incorporated herein in its entirety. Variations on any of these
methods are known to or will be readily apparent to those skilled
in the art. It is not intended that this disclosure be limited to
any particular process of manufacture or mode of production.
[0080] Generally, the methods disclosed use compositions that are
formulated for pharmaceutical use. The methods do not include
simple ingestion of a cruciferous vegetable such as broccoli, but
rather include the use of a pharmaceutical composition comprising
sulforaphane in an isolated or synthetic form. Pharmaceutically
acceptable carriers include, but are not limited to, physiological
saline, Ringer's, phosphate solution or buffer, buffered saline,
and other carriers known in the art.
[0081] The route of administration may be one or more of buccal
(via membranes of the mouth), oral, or enteral (via the
gastrointestinal tract). The pharmaceutically acceptable carrier
can also be selected on the basis of the desired route of
administration of SFN. The desired route of administration may be
one or more of buccal, oral, or enteral. For example, in a one
embodiment the carrier is suitable for oral administration. In some
embodiments, the composition includes a carrier or additional agent
that is suitable for promoting delivery of the compound(s) to the
gastrointestinal or intestinal tract.
[0082] Pharmaceutical compositions intended for oral administration
may be prepared according to any method known in the art for the
production of pharmaceutical compositions, and such compositions
may contain one or more agents selected from the group consisting
of sweetening agents, flavoring agents, coloring agents, and
preserving agents in order to provide pharmaceutically suitable and
palatable preparations.
[0083] In an embodiment, methods include administering a
pharmaceutical composition that includes about 50 to about 400
.mu.M of SFN. For example, the methods include administering a
pharmaceutical composition that includes about 50, about 100, or
about 200 .mu.M of SFN. In a further embodiment, the methods
include administering a pharmaceutical composition that includes
about 0.5 to about 10 .mu.M of SFN per kilogram of bodyweight of a
subject. For example, the methods include administering a
pharmaceutical composition that includes about 0.5, about 2.0, or
about 4.0 .mu.M of SFN per kilogram of bodyweight of a subject.
[0084] Oral delivery of the pharmaceutical compositions of the
present disclosure can include compositions, as are well known in
the art, to provide prolonged or sustained delivery of the drug to
the gastrointestinal and/or intestinal tract by any number of
mechanisms. These include, but are not limited to, pH sensitive
release from the dosage form based on the changing pH of the small
intestine, slow erosion of a tablet or capsule, retention in the
stomach based on the physical properties of the composition,
bioadhesion of the dosage form to the mucosal lining of the
intestinal tract, or enzymatic release of the active drug from the
dosage form. For some of the therapeutic compositions, the intended
effect is to extend the time period over which the active drug
molecule is delivered to the site of action by manipulation of the
dosage form. Thus, enteric-coated controlled release compositions
are within the scope of the present disclosure. Suitable enteric
coatings include cellulose acetate phthalate, polyvinylacetate
phthalate, hydroxypropylmethylcellulose phthalate and anionic
polymers of methacrylic acid, and methacrylic acid methyl
ester.
[0085] The SFN that is useful in the present methods is of a
quality and stability suitable for pharmaceutical use. Oral
administration includes buccal or "sub-lingual" administration via
membranes of the mouth. This can be accomplished using lozenges or
a chewable gum containing SFN. The SFN can be deposited in a
flavored base, usually sucrose, acacia, or tragacanth, and
pastilles comprising the SFN in an inert base such as gelatin and
glycerin or sucrose and acacia.
[0086] Multiple forms of SFN are appropriate for oral
administration. In different embodiments, these include but are not
limited to stock SFN provided as a solid (including powders) or a
liquid. Stock SFN may then be further mixed and prepared for
administration.
[0087] Solid dosage forms for oral administration include, but are
not limited to, capsules, tablets, pills, powders, and granules. In
such solid dosage forms, the active compounds are mixed with at
least one pharmaceutically acceptable excipient or carrier such as,
but not limited to, sodium citrate or dicalcium phosphate.
[0088] The pharmaceutical composition can include fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid. Optionally, binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,
sucrose, and acacia can be include. In additional embodiments, the
pharmaceutical compositions include humectants, such as glycerol,
and/or disintegrating agents, such as agar-agar, calcium carbonate,
potato or tapioca starch, alginic acid, certain silicates, and
sodium carbonate. The compositions can also include solution
retarding agents such as paraffin, absorption accelerators such as
quaternary ammonium compounds, wetting agents such as acetyl
alcohol and glycerol mono stearate, absorbents such as kaolin and
bentonite clay, and/or lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate.
[0089] In the case of capsules, tablets and pills, the dosage form
can also comprise buffering agents. Tablets may contain the active
ingredient in admixture with non-toxic pharmaceutically acceptable
excipients, which are suitable for the production of tablets. These
excipients may be, for example, inert diluents, such as calcium
carbonate, sodium carbonate, lactose, calcium phosphate or sodium
phosphate, granulating and disintegrating agents, for example,
maize starch, or alginic acid, binding agents, for example starch,
gelatin or acacia, and lubricating agents, for example magnesium
stearate, stearic acid, or talc.
[0090] The tablets may be uncoated, or they may be coated by known
techniques to delay disintegration and adsorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate may be employed.
[0091] Pharmaceutical compositions suitable for oral administration
can be presented in discrete units each containing a predetermined
amount of at least one therapeutic compound useful in the present
methods; as a powder or granules; as a solution or a suspension in
an aqueous or non-aqueous liquid; or as an oil-in-water or
water-in-oil emulsion. As indicated, such compositions can be
prepared by any suitable method of pharmacy, which includes the
step of bringing into association the active compound(s) and the
carrier (which can constitute one or more accessory ingredients).
In general, the compositions are prepared by uniformly and
intimately admixing the active compound with a liquid or finely
divided solid carrier, or both, and then, if necessary, shaping the
product.
[0092] For example, a tablet can be prepared by compressing or
molding a powder or granules of the compound, optionally with one
or more accessory ingredients. Compressed tablets can be prepared
by compressing, in a suitable machine, the compound in a
free-flowing form, such as a powder or granules optionally mixed
with a binder, lubricant, inert diluent and/or surface
active/dispersing agent(s). Molded tablets can be made by molding,
in a suitable machine, the powdered compound moistened with an
inert liquid diluent.
[0093] Solid compositions of a similar type can also be employed as
fillers in soft and hard filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0094] The solid dosage forms of tablets, dragees, capsules, pills,
and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well known in the
pharmaceutical formulating art. They can optionally contain
opacifying agents and can also be of a composition that they
release the active ingredient(s) in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions which can be used include polymeric
substances and waxes. The active compounds can also be in
micro-encapsulated form, if appropriate, with one or more of the
above mentioned excipients.
[0095] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions, solutions,
suspensions, syrups, teas, and elixirs. In addition to the active
compounds, the liquid dosage forms can contain inert diluents
commonly used in the art such as, for example, water or other
solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethyl formamide, oils (in particular, cottonseed, groundnut,
com, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring,
and perfuming agents. In some embodiments, suspensions, in addition
to the active compounds, can contain suspending agents as, for
example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol
and sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures
thereof.
[0096] A drinkable tea containing SFN can also be used in the
present methods. A drinkable tea may be taken in a liquid form or
in a once pulverized or granulated form together with water or hot
water. When it is in a powdery or granular form, the drinkable tea
may be contained in a cavity of mouth before taking hot water or
water like the conventional powdery or granular drinkable tea, or
it may be taken after once dissolving in hot water or water. One or
more components, such as a sugar, mint, or other flavor, can be
added to improve taste and easiness as a drinkable drug. Teas,
syrups and elixirs can be formulated with sweetening agents, for
example glycerol, sorbitol or sucrose. Such compositions can also
contain a demulcent, a preservative, and flavoring and coloring
agents.
[0097] SFN can be administered in conjugation with radiation
therapy and/or another agent such as interferon (including
high-dose interferon), dacarbazine, IL-2 or other cytokine, BCG
vaccine, imiquimod, ipilimumab, vemurafenib and temozolomide. In
particular, the pharmaceutical compositions of the present
disclosure, or compositions in which they are included, can be
administered orally, for example, as tablets, coated tablets,
dragees, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions, hard or soft capsules,
or syrups, teas, or elixirs. Compositions intended for oral use may
be prepared according to any method known in the art for the
manufacture of pharmaceutical compositions and such compositions
may contain one or more agents selected from the group consisting
of sweetening agents, flavoring agents, coloring agents and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations.
[0098] Compositions for oral use may also be presented as hard
gelatin capsules wherein the active ingredients are mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredients are present as such, or mixed with water or an oil
medium, for example, peanut oil, liquid paraffin, any of a variety
of herbal extracts, milk, olive oil, or other plant oil.
[0099] Aqueous suspensions can be produced that contain the active
materials in admixture with excipients suitable for the production
of aqueous suspensions. Such excipients are suspending agents, for
example, sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethyl-cellulose, sodium alginate,
polyvinylpyrrolidone gum tragacanth and 30 gum acacia; dispersing
or wetting agents may be naturally occurring phosphatides, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethyleneoxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example
polyoxyethylene sorbitan monooleate.
[0100] The aqueous suspensions may also contain one or more
preservatives, for example, ethyl or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, or one
or more sweetening agents, such as sucrose or saccharin.
[0101] Oily suspensions may be formulated by suspending the active
ingredients in an omega-3 fatty acid, a vegetable or plant oil, for
example arachis oil, olive oil, sesame oil, broccoli oil, or
coconut oil, or in a mineral oil such as liquid paraffin. The oily
suspensions may contain a thickening agent, for example beeswax,
hard paraffin or cetyl alcohol.
[0102] Sweetening agents, such as those set forth above, and
flavoring agents may be added to provide a palatable oral
preparation. These compositions may be preserved by the addition of
an antioxidant such as ascorbic acid.
[0103] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent, a
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0104] The present disclosure may also include safe and effective
amounts of isotonicity agents, e.g., salts, such as sodium
chloride, or non-electrolyte isotonicity agents such as sorbitol,
and mannitol.
[0105] The solubility of the components of the present compositions
may be enhanced by a surfactant or other appropriate co-solvent in
the composition. Such co-solvents include polysorbate 20, 60, and
80, polyoxyethylene/polyoxypropylene surfactants (e.g., Pluronic
F-68, F-84 and P-103), cyclodextrin, or other agents known to those
skilled in the art. Typically, such co-solvents are employed at a
level of from 0.01% to 2% by weight.
[0106] Pharmaceutically acceptable excipients and carriers
encompass all the foregoing and the like. The above considerations
concerning effective compositions and administration procedures are
well known in the art and are described in standard textbooks. See
e.g. Gennaro, A. R., Remington: The Science and Practice of
Pharmacy, 15th Edition, (Lippincott, Williams and Wilkins), 2000;
Hoover, John E., Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1975; Liberman et al., Eds.,
Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980;
and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd
Ed.), American Pharmaceutical Association, Washington, 1999.
Methods for Reducing the Progression of Atypical Nevi and
Preventing Melanoma
[0107] Methods are provided for preventing melanoma in a subject.
The methods include selecting a subject at risk of developing
melanoma, and administering orally to the subject a therapeutically
effective amount of a pharmaceutical composition comprising an
effective amount of SFN, thereby preventing melanoma from
developing in the subject. Thus, in several embodiments, the
methods can prevent the appearance of melanoma precursor lesions
and the appearance of superficial spreading melanoma, nodular
melanoma, acral lentiginous melanoma, lentigo maligna melanoma, and
other melanomas.
[0108] In some non-limiting examples, the methods reduce the number
of new atypical nevi appearing and/or of reduce the progression of
nevi from a less to a more atypical type. Thus, reducing
progression includes, but is not limited to, reducing emergence of
additional nevi and/or reducing the conversion of atypical nevi to
more atypical types.
[0109] In some embodiments, subjects are selected because they are
at increased risk of developing melanoma. Increased risk for
melanoma can be defined in comparison to the general population as
a history of atypical nevi, a history of dysplastic nevi syndrome,
a history of melanoma, a genotype including a loss of function
mutation in an MC1R gene, and/or other indicators of increased risk
of melanoma. In some embodiments, subjects are selected that have a
loss of function mutation in an MC1R gene and/or an immediate or
extended, living or deceased family member having this mutation.
See US Patent Application Publication No. 2011/0201011, which is
hereby incorporated by reference in its entirety, for methods of
selecting for a subject with a loss of function mutation in an MC1R
gene. In other embodiments, a subject is selected that has a
relatively high number of atypical nevi and/or a high degree of
atypia among these nevi. In another embodiment, a subject can be
selected that has a history of UV exposure to the skin. Any of
these can be a condition for selecting subjects with increased risk
of developing melanoma.
[0110] Risk factors for melanoma can be ascertained through
clinical interviews, and/or through clinical testing, and/or
through subjects reporting via questionnaires, and/or through
epidemiological data indicating an at-risk population, and/or any
other means. Clinical interviews and questionnaires can be
conducted in accordance with accepted clinical practice known to
those in the medical field. Clinical testing can be conducted in
accordance with accepted clinical practice known to those in the
medical field and can include genetic testing for loss of function
mutations in MC1R genes. MC1R testing can involve genotyping from
harvested saliva samples of living subjects or their relatives.
[0111] The presence of melanoma and the number and degree of atypia
of nevi may be determined by any of various visual means, including
but not limited to visual inspection, such as with the naked eye,
with the aid of an optical device such as a dermatoscope,
inspection of analog or digital photographs or video of a subject's
skin, or the application of other techniques known in the field of
dermatoscopy. Visual inspection can be enhanced by the use of dyes
and contrast agents. The presence and type of melanoma or a nevi
can also be assessed using biopsies and histological methods known
in the medical field. Any of these techniques may be applied at
selected locations on the body, and/or they may be part of a whole
body screening. The presence of nevi reaching 4 or more millimeters
in diameter can indicate increased risk of melanoma compared with
smaller nevi. In some embodiments, smaller nevi may be a risk
indicator. The presence of one or more, or, in some embodiments,
two or more nevi reaching 4 or more millimeters in diameter can
indicate increased risk of melanoma compared with smaller nevi. The
presence of two or more nevi, with one or more of those nevi having
been classified as atypical on a tiered scale, can indicate
increased risk of melanoma compared with a lack of atypical nevi.
Thus, fewer subjects can be selected for treatment using the
methods disclosed here.
[0112] In some embodiments, the degree of atypia can be assessed
morphologically and/or visually and/or photographically on a tiered
scale, with successive tiers used to classify lesser and greater
degrees of atypia. In an embodiment, clinical assessment of atypia
may be mild, moderate, and severely atypical. In another
embodiment, a tiered scale may have tiers, for example, labeled "A"
through "F," with "F" indicating a least atypical nevus, and with
"A" indicating a most atypical nevus. In an embodiment nevi are
classified based on observed properties of a nevi, including size,
shape/asymmetry/roundness/presence or projections,
continuity/smoothness/irregularity of border,
color/pigmentation/variegation, degree raised above skin surface,
firmness, continuity vs. broken surface structure, the presence of
other structural features or abnormalities, and known evolution
over time. In an embodiment, histopathological assessment of atypia
may be mild, moderate, and severe dysplasia (atypia).
[0113] The methods include administering a therapeutically
effective amount of a pharmaceutical composition including SFN to
the subject, wherein the pharmaceutical composition is administered
orally. Any of the pharmaceutical compositions disclosed herein can
be used in these methods. In specific non-limiting examples, the
pharmaceutical composition is a tea or a gel capsule.
[0114] In an embodiment, methods include administering a
pharmaceutical composition that includes about 50 to about 400
.mu.M of SFN. For example, the methods include administering a
pharmaceutical composition that includes about 50, about 100, or
about 200 .mu.M of SFN. In a further embodiment, the methods
include administering a pharmaceutical composition that includes
about 0.5 to about 10 .mu.M of SFN per kilogram of bodyweight of a
subject. For example, the methods include administering a
pharmaceutical composition that includes about 0.5, about 2.0, or
about 4.0 .mu.M of SFN per kilogram of bodyweight of a subject.
[0115] The pharmaceutical composition can be administered at
selected time intervals over a treatment course of selected
duration. The pharmaceutical composition can be administered thrice
daily, twice daily, daily, bi-weekly, weekly or monthly. The
subject can be treated over an extended period, such as for weeks,
months or years. Specific examples include, but are not limited to,
administration for a week, about ten days, about two weeks, about a
month, about three months, about six months, about nine months,
about a year, about two years, about three years, about five years,
about ten years, or for the remaining lifespan of the subject.
[0116] In some embodiments, the effectiveness of the administration
of SFN can be monitored by testing for the expression of STAT1
and/or STAT3 in a biological sample obtained from the subject. In
an embodiment, testing for the expression of STAT1 or STAT3 in
biopsy samples is accomplished using an immunoassay or an
immunohistological assay, for example using antibodies to either or
both of STAT1 and STAT3, where antibodies to both are known in the
field and commercially available. In an embodiment, the sample in
which STAT expression is monitored is tissue from a biopsy, for
example a biopsy of normal skin or a biopsy of a nevus.
[0117] In one embodiment, nucleic acid based methods are utilized.
These methods include serial analysis of gene expression (SAGE
techniques), RT-PCR, quantitative PCR, real time PCR, Northern
blot, dot blots, microarrays, amongst others. Generally, with
regard to nucleic acids, any method can be utilized provided it can
detect the expression of target gene mRNA (STAT1 and/or STAT3) as
compared to a control. In other embodiments, the amount of STAT1
and/or STAT3 polypeptides is measured. This can be accomplished
using immunoassays or using spectrometric methods. Both monoclonal
and polyclonal antibodies, and fragments thereof, can also be
utilized to detect and quantify the expression of STAT1 and STAT3.
This can be accomplished, for example, by immunohistochemistry,
immunoassay (such as enzyme-linked immunosorbent assay (ELISA) or
radioimmunoassay (RIA)), Western blotting, flow cytometric or
fluorimetric detection. The antibodies (or fragments thereof) can
be employed histologically, as in immunofluorescence or
immunoelectron microscopy, for in situ detection of STAT1 and/or
STAT3. In situ detection includes contacting a histological
specimen from a subject with labeled antibody, and detecting
binding of the antibody to monocytes within the sample. A wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0118] One of skill in the art can readily identify an appropriate
control, such as a sample from a subject known not to have a
melanoma or atypical nevi (a negative control), a sample from a
subject known to have melanoma (a positive control), or a known
amount of nucleic acid encoding STAT1 and/or STAT3 (a standard or a
normal level found in a healthy subject).
[0119] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
Example 1
SFN's Effect on Melanoma Cells In Vitro
[0120] This example illustrates that sulforaphane (SFN) modulates
growth of cells (SK-MEL31 and SK-MEL37), Cyclin B1, Cdk1, and
Cdc25.
[0121] It was demonstrated that SFN inhibits melanoma cell growth
in vitro. FIG. 1 shows the effect of SFN on SK-MEL31 and SK-MEL37
cell viability as determined through a trypan blue dye exclusion
assay. 4,000 cells were plated in 6-well plates, allowed to attach
overnight, and exposed to different concentrations of SFN for 24,
48, or 72 h at 37.degree. C. A stock solution of SFN was prepared
in DMSO, and an equal volume of DMSO was added to the controls.
Data are mean +/-standard error (SE) of 3 determinations. Similar
results were observed in replicate experiments.
[0122] It was also demonstrated that SFN inhibits melanoma cell
Cyclin B1, Cdk1, and Cdc25 in vitro. FIG. 2 shows immunoblotting
for cyclinB1, Cdk1, Cdc25C and Ser-216 phosphorylated Cdc25C using
lysates from SK-MEL31 cells treated with 20 .mu.M SFN for the
indicated time periods. FIG. 2 also shows immunoblotting for p-Chk1
(Ser-317 phosphorylated Chk1), total Chk1, p-Chk2 (Thr-68
phosphorylated Chk2) and total Chk2 using lysates from SK-MEL31
cells treated with 20 .mu.M SFN for the indicated time periods. The
blots were stripped and re-probed with anti-actin antibody to
normalize for differences in protein loading.
Example 2
SFN's Effect on Hu Melanoma Xenotransplants
[0123] As shown in FIG. 3, D,L-SFN administered orally to SCID mice
lowered STAT3 expression in human melanoma cells transplanted into
the mice.
Example 3
SFN Induces Apoptosis in Human Melanoma Cells SK-MEL 31 and Retards
Growth of SK-MEL 31 Xenografts In Vivo
[0124] This example provides materials and methods for Examples
4-10. Examples 4-10 illustrate that SN increased apoptosis of human
melanoma cells. SFN inhibited proliferation of cultured SK-MEL 31
cells by inducing apoptosis characterized by appearance of cells
with sub-G0/G1 DNA content, cleavage of poly (ADP-ribose)
polymerase (PARP), formation of cytoplasmic histone associated DNA
fragments, and condensed nucleus. Oral administration of SFN
significantly retarded the growth of SK-MEL 31 xenografts in nude
mice, accompanied by increase of apoptotic bodies in SFN treated
tumor specimen by TUNEL assay. SFN induced apoptosis was associated
with down regulation of Bcl-XL, XIAP and cIAP1, release of
cytochrome-c and Smac/DIABLO into the cytoplasm and activation of
caspas-8, -9 and -3. Introduction of general and specific caspase
inhibitors blocked the activation of caspase-3 and subsequent
apoptosis after SFN exposure. These data show the pro-apoptotic
effect of SFN against human melanoma both in vitro and in vivo.
[0125] D,L-SFN (>99% pure) was purchased from LKT Laboratories
(St Paul, Minn.), and dissolved in DMSO before use. RPMI 1640,
penicillin/streptomycin antibiotic mixture, and fetal bovine serum
were from Gibco (Grand Island, N.Y.), propidium iodide and
4',6'-diamidino-2-phenylindole-dihydrochloride (DAPI) were from
Sigma (St Louis, Mo.). LIPOFECTAMINE 2000.TM. and G418 were
purchased from Invitrogen (Carlsbad, Calif.). RNaseA was purchased
from Promega (Madison, Wis.), and the reagents for electrophoresis
were from Bio-Rad (Hercules, Calif.). Antibodies for Bcl-XL, Bak
and cytochrome c were purchased from Santa Cruz Biotechnology
(Santa Cruz, Calif.), antibodies specific for Bax, Smac/DIABLO,
cleaved caspase-3 and cleaved caspase-8, cleaved caspase-9, cIAP1
and poly (ADP-ribose) polymerase (PARP) were purchased from Cell
Signaling Technology (Beverly, Mass.), anti Bcl-2 antibody was
purchased from Dakocytomation (Denmark), anti XIAP (X-linked
inhibitor of apoptosis)antibody was purchased from BD PharMingen
(San Diego, Calif.). Anti actin antibody was purchased from
Oncogene Research Products (Boston, Mass.). The caspase inhibitors
z-VAD fmk (general caspase inhibitor), z-IETD fmk (caspase-8
specific inhibitor) and z-LEHD fmk (caspase-9 specific inhibitor)
were purchased from MP Biochemicals (Aurora, Ohio). The caspase-8
and caspase-9 colorimetric assay kits were purchased from R&D
systems (Minneapolis, Minn.). Cytoplasmic histone associated DNA
fragments detection ELISA kit was purchased from Roche Diagnostics
GmbH (Mannheim, Germany). ProteoExtract.TM. Subcellular Proteome
Extraction kit was purchased from Calbiochem (San Diego,
Calif.).
Cell Culture and Cell Proliferation Assays
[0126] Monolayer cultures of SK-MEL 31 cells were maintained at
37.degree. C. in RPMI 1640 supplemented with 10% (v/v) heat
inactivated fetal bovine serum and antibiotics in a humidified
atmosphere of 5% CO2 and 95% air. Trypan blue dye exclusion assay
was used to study the effect of SFN on proliferation of SK-MEL 31
cells. SK-MEL 31 cells were seeded in 12 well plates in triplicate
at 1.times.10.sup.4/well and allow to attach overnight. Cells were
then incubated with Sulforaphane (0, 10, 20, 40 .mu.M) for 24, 48
and 72 hours. Both floating and adherent cells were pelleted by
centrifugation, and resuspended with 0.4% Trypan blue, stained and
unstained cells were counted for each sample. To evaluate the
effect of SFN treatment on cell cycle progression, SK-MEL 31 cells
(5.times.10.sup.5 cells) were seeded in triplicate in T25 flasks,
and allowed to attach overnight. The media was replaced with fresh
complete media containing 20 .mu.M SFN or equal volume of DMSO as
control. After 24 hours of incubation, floating and attached cells
were collected, washed with PBS and fixed with 70% ethanol. Fixed
cells were then incubated with 80 mg/ml RNase A and 50 mg/ml
propidium iodide for 30 minutes, and DNA content for cell cycle
analysis quantified was analyzed using a Coulter Epics XL Flow
Cytometer.
Transfection of SK-MEL 31 Cells with pSFFV-Bcl-XL Plasmid DNA
[0127] To establish Bcl-XL over-expression cells, SK-MEL 31 cells
were transfected with pSFFV-Bcl-XL or pSFFV-neo plasmids at 1:2
ration (.mu.g plasmid DNA: .mu.l Lipofectamine) using LIPOFECTAMINE
2000.TM.. Transiently transfected cells were selected and
maintained in RPMI 1640 containing 300 .mu.g/ml of G418 until
experimentation.
Determination of Apoptosis
[0128] Apoptosis induction in SFN-treated SK-MEL 31 cells was
assessed by (i) quantification of cytoplasmic histone associated
DNA fragments, (ii) Western blot analysis for PARP cleavage and
(iii) DAPI dye for nuclear morphology assay. For quantification of
cytoplasmic histone-associated DNA fraction, SK-MEL 31 cells were
seeded at 4.times.10.sup.3 cells/well in 96-well plate and allowed
to attach overnight. The media was replaced with fresh complete
media containing 0, 2.5, 5, 10 .mu.M of SFN. After 24 hours, the
floating and attached cells were pelleted and lysed with lysis
buffer supplied with the cell death detection ELISA kit, lysates
were assayed for apoptosis according to the manual. SK-MEL 31 cells
were seeded at 1.times.10.sup.4 cells/well on a round glass
coverslip in 12 well plates. Cells were then incubated for 24 hours
with 0, 10, or 20 .mu.M SFN, attached cells were fixed with 3%
paraformaldehyde in PBS, permeabilized with 1% TRITON.RTM. X-100,
stained with DAPI dye and inspected under a fluorescent microscope
after stained with DAPI.
Western Blot Analysis
[0129] 2.times.10.sup.6 SK-MEL 31 cells were seeded into T75
flasks, after treatment with 20 .mu.M SFN or equal volume of DMSO
(control) for specified time interval; floating and attached cells
were collected and lysed with TRITON.RTM. X-100/SDS lysis buffer at
4.degree. C. for 30 minutes. Cell lysate was cleared by
centrifugation at 16000 rpm for 15 min Lysate proteins were
resolved by SDS-PAGE gel electrophoresis and transferred onto
nitrocellulose membrane. The membrane was immersed into a phosphate
buffered saline (PBS) solution containing 0.2% TWEEN.RTM. 20 and 5%
(w/v) non-fat dry milk, and then incubated with desired primary
antibody for 1 hour at room temperature. After incubation with
appropriate secondary antibody, the immunoreactive bands were
visualized using enhanced chemiluminescence method. The conjugated
horseradish peroxidase (HRP) secondary antibody was deactivated
with sodium azide, then membranes were re-probed with anti-actin
antibody to standardize protein loading. For cytochrome c and
Smac/DIABLO, the cytosolic fractions were prepared with the
PROTEOEXTRACT.TM. Subcellular Proteome Extraction kit according to
the manufacturer's instructions and Western blot analysis were
performed as described above.
Xenograft Assay
[0130] Female athymic mice (6-week-old) were purchased from Taconic
(Germantown, N.Y.). SK-MEL 31 cells were suspended in
pharmacological grade normal saline, and 0.1 ml suspension
containing 5.times.10.sup.6 cells was injected subcutaneously on
right flank of each mouse. Mice were randomized into two groups of
10 mice per group. Experimental animals were gavaged with SFN (5.6
.mu.mol SFN in 0.1 ml normal saline) three times per week (Monday,
Wednesday and Friday) beginning two weeks before tumor
implantation. Control mice received an equal volume of the vehicle.
Tumor volume was determined based on measurement of the length and
width of the tumor, V=1/2.times.length.times.width.sup.2.
Statistical significance of difference in tumor volume was assessed
by Student's t-test. Upon the termination of the experiment, the
tumor tissues were harvested and processed for analysis of
apoptotic bodies by immunohistochemistry method. Tumor tissues were
embedded in paraffin, sectioned (6 .mu.m), de-paraffinized and
processed for determination of apoptotic bodies using APOTAG.RTM.
Plus Peroxidase In Situ Apoptosis detection kit (Intergen, N.Y.)
according to the manufacturer's instructions. Brown color apoptotic
bodies in tumor sections of control and SFN-treated group were
counted by two pathologists independently and blindly under a
microscope at 400.times. magnification. Four randomly selected
fields on each tumor section were counted for apoptotic bodies.
Example 4
SFN Inhibits Proliferation of SK-MEL 31 Cells In Culture
[0131] The effect of SFN on proliferation of SK-MEL 31 cells was
determined by trypan blue dye exclusion assays and flow cytometric
analysis of cellular DNA content. All the assays were carried out
in two independent experiments, and the results from a
representative experiment are shown in FIG. 4. SFN treatment caused
a statistically significant decrease in number of viable cells in
trypan blue dye exclusion assay, p<0.05 (FIG. 4A). At a 24 hour
time point, the number of viable cells exposed to 10, 20 and 40
.mu.M SFN was reduced to 58%, 46% and 26% compared with control,
respectively. Even greater effects appeared following 48 or 72
hours exposure to SFN at all the concentrations. The number of
viable cells exposed to 10, 20 and 40 .mu.M SFN was reduced to 27%,
23% and 10% compared to control at 48 hour time point, and to 19%,
13% and 10% at 72 hour time point (p<0.05). The cell cycle
distribution after SFN exposure showed significantly increase of
Sub-G.sub.0/G.sub.1 and G2M phase and decreased G.sub.0/G.sub.1
phase cell number (p<0.05), suggesting both apoptosis and cell
cycle arrest at G2M phase (FIG. 4B).
Example 5
SFN Induces Apoptosis in SK-MEL 31 Cells
[0132] The FACS assay suggested there was apoptosis following SFN
exposure in SK-MEL 31 cells, so further experiments were carried
out to verify this effect. Apoptosis occurs with a number of
morphological and cellular changes, such as membrane blebbing,
chromatin condensation, DNA fragmentation, and key cellular
proteins like PARP cleavage. Pro-apoptotic effect of SFN was
assessed by ELISA based quantification of cytoplasmic histone
associated DNA fragments. Incubation with SFN for 24 hours resulted
in a dose-dependent and statistically significant increase in
cytoplasmic histone associated DNA fragments. Exposure to 5 and 10
.mu.M SFN resulted in .about.4.7- and .about.11.4-fold increase in
the levels of cytoplasmic histone associated DNA fragments compared
with DMSO treated control (p<0.01, FIG. 5A). Western blot assay
showed there was an obvious increase of PARP cleavage from 16 to 24
hour after SFN treatment (FIG. 5B). DAPI dye assay was used to
detect condensed nuclear, the characteristic morphology change of
apoptosis. (FIG. 5C).
Example 6
Orally Administered SFN Retards Growth of SK-MEL 31 Xenografts In
Vivo
[0133] In vivo studies were conducted to testify whether SFN
administration affects growth of SK-MEL 31 xenografts in nude mice.
Effect of oral administrated SFN on SK-MEL 31 xenografts growth was
determined in two independent experiments. SFN treatment caused a
significant inhibition of SK-MEL 31 xenograft growth. At 16 days
after tumor implantation, the average tumor volumes in control and
SFN-treated mice were 310.5.+-.113.3 and 154.4.+-.95.4 mm.sup.3
respectively, reflecting a 50% reduction in tumor volume in SFN
group (p<0.01, FIG. 6A). Similar statistically significant
inhibition of SK-MEL 31 tumor xenograft growth after oral
administration of SFN was observed in the second independent
experiment. Tumor wet weight comparison showed significant decrease
in the SFN treated group (p<0.01, FIG. 6B). TUNEL assay revealed
.about.4.4 fold higher count of apoptotic bodies in tumor sections
from SFN-treated mice than in controls (p<0.05, FIG. 6C).
Example 7
Bcl-2 Family Protein Level Changes in SFN-Induced Apoptosis
[0134] To explore the mechanisms of SFN induced apoptosis in SK-MEL
31 cells, the effect of SFN treatment on expression of Bcl-2 family
of anti-(Bcl-2 and Bcl-XL) and pro-apoptotic (Bax and Bak) proteins
was determined. Representative Western blots illustrating the
effect of SFN treatment on Bcl-2 family protein level are shown in
FIG. 7. Exposure of SK-MEL 31 cells to 20 .mu.M SFN resulted in
down-regulation of Bcl-XL, that was evident from the 2 hour time
point (FIG. 7A). No marked change of Bcl-2 was identified, and Bax
and Bak expression was observed at all time points
investigated.
Example 8
Involvement of Caspases in SFN-Induced Apoptosis
[0135] Caspases are aspartate-specific cysteine proteases that play
critical roles in apoptosis. Activation of caspases results in
cleavage and inactivation of key cellular proteins, including the
DNA repair enzyme PARP. Activation of caspases after SFN treatment
was evaluated by immunoblot for cleaved caspase-8, -9 and
caspase-3, and the caspase-8, 9 activity changes with colormetric
assay kits. Treatment of SK-MEL 31 cells with 20 .mu.M SFN resulted
in cleavage of procaspase-9, procaspase-8 and procaspase-3 as
evidenced by appearance of intermediates (37 KDa for caspase-9,
43/41 KDa for caspase-8 and 19/17 KDa for caspase-3) at the 24 hour
time point (FIG. 8C). Treatment with 20 .mu.M SFN significantly
induced a caspase-8 activity increase, specifically 45% (16 hour)
and 73% (24 hour) increase compared with the control group, with a
148% increase in caspase-9 activity at 24 hours (p<0.05, FIGS.
8A-8B). To confirm the involvement of caspases in the SFN induced
apoptosis in SK-MEL 31 cells, inhibitors for general caspase
(z-VADfmk) and caspase-8 (z-IETDfmk), caspase-9 (z-LEHDfmk) were
introduced to block the corresponding caspase activity. Incubation
with the caspase inhibitors significantly lowered the SFN induced
cytoplasmic histone associated DNA fragments level in Cell Death
ELISA assay (p<0.05, FIG. 8D). Western blot of cleaved caspase-3
showed blockage of caspase-3 cleavage after introduction of caspase
inhibitors before SFN exposure (FIG. 8E).
Example 9
Other Apoptosis Regulating Proteins Involved
[0136] There are many proteins other than caspases that take part
in apoptosis. Some of these protein level changes after SFN
exposure were determined to examine their role in SK-MEL 31 cell
apoptosis. IAPs are a group of proteins that exert inhibitory
effect on cellular apoptosis through different pathways, and
treatment with SFN caused consecutive cIAP1 and XIAP level
decreases with time increasing (FIG. 7B). Smac/DIABLO and
cytochrome c are small molecules released from mitochondria upon
the disruption of the mitochondrial membrane integrity, which exert
their pro-apoptic activity through interaction with other
molecules. The cytosolic levels of Smac and cytochrome c showed
time-dependent increases after SFN incubation, with significantly
increases occurring after 8 hours for both Smac and cytochrome c
(FIG. 9E).
Example 10
The Protective Role of Bcl-XL Against SFN Induced Apoptosis
[0137] It was shown consistently that Bcl-XL was down-regulated
after exposure to SFN. To determine the exact role of Bcl-XL in SFN
induced apoptosis, SK-MEL 31 cells were transiently transfected
with pSFFV-Bcl-XL and pSFFV-neo (vector), and assayed for the
effect of Bcl-XL over-expression on SFN induced apoptosis and
associated molecular changes. FIG. 9A shows the increased level of
Bcl-XL in pSFFV-Bcl-XL transfected cells, but not in the vector
transfected cells. Over-expression of Bcl-XL partially blocked the
apoptosis induced by SFN (p<0.01, FIGS. 9B-9C). This effect was
associated with decreased release of Smac and cytochrome-c into
cytoplasm (FIG. 9E), and decreased activation of caspase-9 and -3
(FIG. 9D).
[0138] The data presented here showed that SFN efficiently
suppressed the proliferation of SK-MEL 31 human melanoma cells in
culture and also in animal experiments. Oral administration of SFN
also retarded the growth of SK-MEL 31 xenografts in nude mice
significantly. No side effect was observed in mice (no obvious
weight loss, or organ weight difference between treated groups) at
the doses used.
[0139] The current results indicate that apoptosis induction is a
major mechanism of the anti-proliferative effect of SFN both in
cultured SK-MEL 31 cells and SK-MEL 31 xenograft in nude mice.
Apoptosis is one of the most potent defenses against cancer, and it
is often deregulated in cancer, which makes it a good target for
cancer prevention. Without being bound by theory, an apoptosis
inducing mechanism for SFN in SK-MEL 31 is shown in FIG. 10.
Traditionally, apoptotic signaling pathways have been divided into
the death receptor-mediated extrinsic pathway and
mitochondria-mediated intrinsic pathway. Both were shown to be
involved in the SFN induced apoptosis in SK-MEL 31 cells. It was
also found that SFN induced apoptosis is associated with
down-regulation of anti-apoptotic Bcl-2 family members: Bcl-XL, but
not Bcl-2, and no significant level changes of Bax and Bak were
observed, which are pro-apoptotic Bcl-2 family members. Without
being bound by theory, the release of cytochrome c and Smac
suggests the active involvement of mitochondria in apoptosis
induction, while the decrease of XIAP and cIAP1 level at an early
time point suggests their inhibitory effects were abrogated to
facilitate apoptosis. See Li et al., Cell (1997) 91: 479-489; Du et
al., Cell (2000) 102: 33-42; and Deveraux et al., EMBO (1998) 17:
2215-2223. Even though no up-regulation of Bax and Bak was
observed, the consistent down-regulation of Bcl-XL and the fact
that over-expression of Bcl-XL protected against SFN induced
apoptosis suggest that the increased ratio of pro-/anti-apoptotic
molecules resulted in activation of mitochondrial pathway. These
changes form a complete caspase-activating cascade, leading to the
activation of initiator caspase (caspase-9) and executioner caspase
(caspase-3) and subsequent apoptotic changes (cleavage of PARP and
appearance of cytoplasmic histone associated DNA fragments and
condensed nucleus). See Thornberry et al., Science (1998) 281:
1312- 1316; and Wolf et al., J. Biol. Chem. (1999) 274:
20049-20052. The cleavage of procaspase-8 and apoptosis blocking
effect of caspase-8 specific inhibitor suggest the active role of
caspase-8 mediated pathway in SFN induced apoptosis in human
melanoma cells. This also indicated that there was significant G2/M
phase cell cycle arrest, as shown by the FACS assay of cellular DNA
content, which also account for the proliferation inhibition both
in vitro and in vivo.
[0140] In conclusion, cultured SK-MEL 31 human melanoma cell and
xenograft in nude mice are highly sensitive to growth inhibition by
SFN, and apoptosis induction is one of the key mechanisms for this
effect.
Example 11
SFN's Effect on Progression of Atypia In Nevi, A Clinical Trial
[0141] Sulforaphane (SFN) modulates progression of atypia in nevi.
SFN affects the expression of STAT proteins in melanocytic and
stromal elements of atypical nevi, which are precursor lesions and
risk markers of melanoma. In this exemplary protocol, eighteen
individuals in total receive oral SFN (SFN) standardized for 3
different concentrations of actual SFN content. Three groups of six
subjects are randomly assigned to receive oral SFN at dosages of 50
.mu.M, 100 .mu.M, or 200 .mu.M daily. Due to the established safety
of SFN at all of the relevant dosage levels, no lower dosage level
is completed prior to escalating to the next higher dosage level;
i.e., subjects are randomized across all of the SFN dosage
levels.
Eligibility Criteria
[0142] Subjects have at least two atypical nevi of .gtoreq.4 mm
diameter and prior diagnosis of melanoma. [0143] Subjects are
.gtoreq.age 12. [0144] Subjects do not have known allergies to
cruciferous vegetables. [0145] Subjects agree to abstain from
dietary sources of glucosinolates and isothiocyanates beginning
three days prior to the protocol and throughout duration of the
active protocol (28 days). [0146] Subjects are non-smokers for at
least 6 months as smoking is found to interfere with the
measurement of SFN metabolites. [0147] Female subjects are not
pregnant or breast feeding prior to and during course of the
protocol. [0148] Subjects meet Grade I criteria for complete blood
count and complete metabolic panel as specified in the Common
Toxicity Criteria (CTC) established by the National Cancer
Institute (NCI) to be eligible for the protocol.
Protocol
[0149] The protocol is conducted as follows and as shown in FIG.
11: [0150] Identify 2-6 atypical nevi from eligible individuals and
label "A-F" based on degree of atypia. [0151] Obtain biopsy
(excisional or large punch) of an atypical nevus by random
selection at protocol onset to act as control and assess the degree
of atypia by histopathological and photographic examination and
evaluate the levels of STAT 1, STAT3 at baseline prior to SFN
administration. Obtain an adjacent tissue biopsy of normal skin and
plasma sample at protocol onset to establish baseline SFN levels.
[0152] SFN is orally administered to the protocol population in 3
randomly assigned dosage groups (50, 100, and 200 .mu.M daily) for
28 days (4 weeks), as shown in FIG. 11. Following 27 days of SFN
administration (i.e., protocol day 28), plasma samples, photography
of index designated atypical nevi, and biopsy of selected atypical
nevi and surrounding normal skin are obtained, as shown in FIG. 12.
These data provide for assessment of: [0153] Concentration of SFN
in normal skin through mass-spectrometric analysis. [0154] Degree
of atypia in nevi through histopathologic and photograph analysis.
[0155] Level of STAT 1 and STAT3 expression within atypical nevi.
[0156] For individuals with .gtoreq.3 atypical nevi an additional
atypical nevus is biopsied and analyzed in similar fashion as above
on day 2 of the protocol, 24 hours following first administration
of BSE-SFN. [0157] The three dosing groups are analyzed for: [0158]
Any change in the clinical or histological features of atypia
observed when comparing pre- and post-SFN administration nevi.
[0159] Optimal dose of SFN to be utilized based on SFN
concentrations measured within normal skin and plasma and effects
on STAT expression levels and nevus histology. [0160] The
modulation of STAT expression levels between control
(pre-administration) and post-SFN-administration nevi in each
group.
Exam and Lesion Selection
[0161] An entry clinical exam is performed within two weeks of
enrollment in the protocol, and the clinical features and number of
nevi are recorded by anatomic site. Any nevi or lesions with
possible question of incipient melanoma are removed prior to the
protocol. Prior to drug administration up to six atypical nevi
.gtoreq.4 mm are photographed and ranked morphologically (visually)
for atypia, with "A" being the most atypical and "F" the least
atypical. Analysis of nevi varies depending on if an individual has
2, 3, or .gtoreq.4 atypical nevi available for analysis as
follows:
[0162] 1. Individuals with 2 Atypical Nevi
[0163] One nevus is randomly selected for photographic
documentation and removal for histological and molecular evaluation
prior to SFN administration and serves as a control. The second
nevus is marked for follow-up photography and biopsy evaluation,
and photographed (or tattooed with India ink) for localization in
the event of clinical involution or change after 28 days of SFN
administration. After 28 days of SFN administration the second
nevus is biopsied to perform routine histological as well as STAT
and SFN expression and immunohistological studies.
[0164] 2. Individuals with 3 Atypical Nevi
[0165] With the addition of a third atypical nevus, that nevus is
biopsied at 24 hours following first administration of oral SFN and
subsequent histological and molecular evaluation similar to nevus
biopsied at day 28.
[0166] 3. Individuals with 4 or More Atypical Nevi
[0167] The remaining nevi (one, two or three nevi) designated to be
analyzed are evaluated at scheduled follow-up visits on day 28, 3
months, and 6 months. Note that these lesions are evaluated
observationally; i.e., they are not biopsied for the purpose of
this clinical investigation.
Photography Session
[0168] Subjects undergo photographic documentation of index nevi
with macrophotography performed upon each nevus using a reference
measurement and white balance tape prior to its removal, as well as
at the start of the protocol. The selected atypical lesions are
photographed for documentation at a fixed magnification of 1:10 and
1:2 under standard lighting conditions. Measurement tapes for size,
date of photograph, and the type of lesion identified are included
on all photographs and also recorded on a flow sheet for reference.
Lesions are identified and coded in a protocol form for evaluating
nevi according to location, symmetry, border, color, size, and
descriptive characteristics. All atypical lesions are photographed
at time of selection and after SFN administration prior to
excision.
[0169] Subjects are asked to have their nevi photographed with the
use of a dermatoscope. The dermatoscope is a small hand-held
optical instrument that is used to visualize skin lesions. It has a
10.times. magnifying lens and uses 6 LED lights for
transillumination. The glass lens is applied directly to the
subject's skin and photographs are taken with an attached digital
camera. No heat or ultraviolet radiation is generated that could
burn or harm the subject. The specific instrument used in this
protocol is the Heine Delta 20 Dermatoscope.
SFN Administration
[0170] A total of 18 subjects are studied with random assignment of
6 individuals to each of three groups that vary according to the
dose of SFN at 50, 100, and 200 .mu.M, respectively. The SFN is
taken once daily at 10 am.+-.2 hours prior to other foods and
fluids. Subjects are asked to fast on day 0 (day prior to biopsy
and SFN administration) and day 27 after midnight in order to limit
the effect food may have on analysis of SFN pharmacokinetics.
Individuals with .gtoreq.3 atypical nevi are asked to fast after
midnight on day 1 as well in preparation for biopsy of an
additional atypical nevus on day 2, 24 hours following first
administration of SFN. On day 1 oral SFN is provided following
biopsy in the clinic. On days 2 (when applicable) and 28 of the
protocol, each subject takes the prescribed dose of oral SFN at 10
am.+-.2 hours in the clinic of the attending oncologist, surgeon or
dermatologist. Biopsies of atypical nevi and biopsies of adjacent
normal skin are obtained at 2 hours.+-.30 minutes.
Follow-up Procedures
[0171] Surveillance photography of atypical nevi for individuals
who had between 4-6 lesions being followed at protocol onset is
performed at 3 and 6 months, as shown in FIG. 12.
Collection of Blood Specimen
[0172] Individuals are required to undergo phlebotomy to assess
pre- and post-administration SFN levels in plasma. On day 1 of the
protocol, a blood sample is drawn prior to administration of oral
SFN to serve as a control for SFN level. A second blood sample is
drawn 2 hours.+-.30 minutes following administration of SFN to
measure SFN concentration within plasma. On day 28, a blood sample
is drawn prior to that day's SFN dose and again 2 hours.+-.30
minutes following the dose to assess SFN concentration in
plasma.
Biopsy of Atypical Nevi and Adjacent Normal Skin
[0173] On days 1 & 2 (when applicable), and day 28 of the
protocol, separate biopsies of designated atypical nevi and
surrounding normal skin are obtained 2 hours.+-.30 minutes
following administration of the morning SFN dose at 10 am.+-.2
hours. Excised atypical nevi are submitted for routine and
investigational histopathologic analysis, with a portion set aside
for immunohistological and other evaluations of STAT signaling and
for evaluation of the molecular and cellular features of each
lesion, while biopsy of normal skin is analyzed for SFN
concentration via mass spectrometry. Normal skin (after collection
to be frozen at -80.degree. C.) is utilized to measure SFN
concentration so that the adjacent nevus is analyzed in its
entirety for the routine and special staining of molecular and
cellular features of interest. Normal skin sample for each
individual is obtained either from the non-melanocytic tail from an
excised atypical nevus or through a second biopsy near the site of
one of the atypical nevi selected at random Immediately upon
removal, nevi are immersed in saline solution and preserved on ice.
Specimens are sent for evaluation. Evaluators examine the lesion
for gross and microscopic features and evaluate the synoptic
factors relevant for atypical nevi; pathologists make the
determination of the amount of tissue that is in excess of that
necessary to establish the histological diagnosis of the lesion.
The remaining tissue, if any, is snap frozen in liquid nitrogen and
is used for molecular and immunologic analyses after completion of
the pathological assessment of the nevus. This remaining tissue is
available to a pathologist if needed for any question in regard to
the routine pathological assessment of the skin lesion.
Analysis of SFN Pharmacokinetics
[0174] At onset on day 1, blood samples are obtained 2 hours.+-.30
minutes following SFN administration and initial measurement of SFN
level within plasma are recorded. Levels should be negligible if
subjects refrained from consuming Brassica containing food and
supplements for three days prior. SFN levels are measured again on
day 28, 2 hours.+-.30 minutes after final administration of oral
SFN.
Measurement of STAT 1 and STAT 3 Expression
[0175] Immunohistochemistry is utilized at the onset of
investigation and for the post-SFN administration samples to assess
levels of STAT1 and STATS expression in control lesions and in nevi
post-SFN exposure on day 28 and also day 2 for individuals with 3
or more atypical nevi. Analysis of the level of STAT expression is
assessed independently by two separate pathologists. The .times.20
objective on microscopy is used to evaluate the whole section in
both pre- and post-administration samples. Pathologists are blinded
to each sample, not knowing ahead of time if it is a pre- or
post-administration sample they are viewing.
[0176] Quantification is decided by consensus of the two
pathologists and a research faculty member. The percentage of cells
with positive staining is evaluated for all samples. Cells with
staining intensity 1+ to 4+ are considered positive; cells without
staining (intensity 0) are considered negative. If the percentage
of stained cells is less than 1%, it is considered zero.
Measurement of Gross Morphologic Response
[0177] Subjects undergo photography at baseline and day 28. Three
investigators evaluate pre- and post-administration photographs in
a blinded manner, to score the features of lesional atypia. Paired
photographs in random blinded order of each nevus from two time
points are evaluated for changes in overall shape and size, color,
border, as well as macular and popular component. Loss of intensity
of pigmentation or features of asymmetry, border
irregularity/diffusion, color variegation, or diminution in size
are taken as signs of reduction of atypia. Nevi revealing increases
in asymmetry, border irregularity/diffusion, color variegation, and
size are deemed to have increased in morphologic atypia. For each
subject, each pair of nevi is compared according to the defining
features: size, asymmetry, color irregularity and border
indistinctness. For each feature, the most atypical nevus of the
two are selected and recorded (with scoring ties indicated). For
individuals with .gtoreq.4 atypical nevi, the three remaining
non-biopsied nevi are followed routinely at day 28, 3 months and 6
months.
Measurement of Histologic Response
[0178] The atypia/dysplasia score of lesions is judged on the
grounds of pre-specified categories of architectural and cytologic
atypia and is determined for lesions removed at the outset of the
protocol and again at the completion of 28 days of oral SFN
administration. The pathologist remains blinded as to the date and
time of administration for scoring evaluations. A portion of the
excised nevus is snap-frozen for future research studies, while the
portion of the specimen deemed necessary for pathological
assessment by the triage pathologist is submitted for routine
histological examination.
Measurement of Molecular Expression/Response
[0179] Preclinical studies indicate that SFN induces apoptosis and
cell cycle arrest in melanoma cells. Therefore, non-exposed and
SFN-exposed nevi are evaluated for evidence of apoptosis (via in
situ TUNEL Assay) and cellular proliferation (via Ki-67
immunostaining). Additionally, alterations in gene expression
profiles of the atypical nevi are assessed through application of
cDNA microarray technology.
Protein Expression Array Analysis
[0180] Biopsy tissues obtained during the course of this protocol,
before and after SFN administration, beyond those required for the
above studies are banked and used for oligonucleotide, DNA, RNA and
protein expression array analysis to explore differences that may
be associated with SFN administration, and the immunological and
molecular features of progression in melanocytic nevi.
[0181] These blood and tumor biopsy samples are stored in a tissue
bank. Samples to be analyzed for expression array differences are
de-identified and are retained.
Drug Composition
[0182] Active Drug Substance: Sulforaphane (SFN) [0183] Other
Names: 1-isothiocyanato-4-(methylsulfinyl)-butane [0184]
Classification: Isothiocyanate [0185] Mode of Action: SFN is an
inhibitor of phase 1 enzymes and leads to reduction in carcinogen
activation due to inhibition of cytochrome P450 dependent
monooxygenases. SFN also is a potent inducer of phase 2 enzymes,
such as glutathione transferases, and leads to accelerated
carcinogen detoxification. Preclinical studies demonstrate that SFN
induces apoptosis and cell cycle arrest in tumor cells. [0186]
Storage and Stability: The prepared oral composition (gel capsules)
of SFN are stored in a freezer at -20.degree. C. [0187]
Preparation: 3-Day-old broccoli sprouts are extracted with hot
water, treated with daikon and freeze dried under good
manufacturing conditions to prepare gel capsule compositions of SFN
containing 50, 100 and 200 .mu.mol of SFN. [0188] Administration:
Daily oral dosing of SFN standardized for SFN at specified dosages
prepared in gel-capsules, provided by Drs. Jed W. Fahey and Paul
Talalay from Johns Hopkins University.
Statistical Considerations
[0189] This protocol evaluates the effects of SFN on atypical nevi.
The objectives of this protocol include evaluating the safety
profile of SFN, quantifying the effects (and the corresponding
variation of the measurements) of oral SFN, and documenting the PK
(pharmacokinetics) and PD (pharmacodynamics) of SFN administered at
different SFN dose levels for subjects with a history of melanoma
and atypical nevi. A total of 18 subjects in 3 cohorts of 6
subjects each with at least two atypical nevi are randomly assigned
to receive oral SFN at SFN doses of 50, 100, or 200 .mu.M daily. Up
to a total of 3 nevi is dissected from the subjects for the
protocol. To ensure that each arm of the study contains roughly the
same number of subjects with various numbers of atypical nevi in
total, the randomization is stratified by the number of atypical
nevi on the skin of the subject (2, 3, or 4 and above). Nevi are
randomized within the same subject to determine which is dissected
before and after SFN administration: [0190] Up to 6 nevi are
studied within the same subject. The nevi are labeled with "A",
"B", "C" to up to "F" based on the morphological measurement of the
severity of disease. A random sequence of the letters is generated
for each subject by the protocol statistician with regard to the
order of the nevi taken out. [0191] For subjects with 2 index
atypical nevi, one is randomly chosen to dissect before the SFN
administration to serve as control and the other nevus to be sent
for analysis. [0192] For subjects with 3 or more nevi, three of
them are randomly chosen and dissected at baseline and at
approximately 24 hours and 28 days. The random order of dissection
is decided by the protocol statistician as disclosed above. The
remaining 1-3 nevi are analyzed by clinical and photographic
analysis at day 28, 3 months, and 6 months. Subsequent evaluations
of these nevi are as per the standard clinical care of these
subjects. [0193] The photographic documentation and measurements of
the atypical nevi and expression of protein markers is measured for
the dissected nevi. The randomization procedure for the order to be
dissected, as disclosed above, ensures that nevi dissected before
and after administration are comparable. The endpoints of the
protocol include: [0194] Photographic outcomes including size of
lesion, border changes, color changes, [0195] STAT1 and STAT 3
protein level measured by IHC, [0196] Other protein marker levels
measured by high-throughput multiplex assays, and [0197]
Pharmacokinetics outcomes at each dose level.
Analysis Plan for Efficacy Endpoints
[0198] The analysis of this protocol focuses on estimation of mean
(and/or median) and variation of the endpoints at both baseline and
post SFN administration dissections.
[0199] The endpoints of interests are either continuous (e.g. nevus
surface size, margin, pigmentation) or ordinal (e.g. IHC measure of
protein level). For continuous variables, the mean (with 95%
confidence interval), median and variance at each time point for
each protocol arm is calculated. Histogram and boxplots are also
used to evaluate the distribution of each variable. The difference
of each variable at a specific post SFN administration time point
from the baseline level is used as the measurement of
investigational treatment effect. Similar descriptive analysis
disclosed above is done for this quantity for each dose group. For
ordinal variables, the range and the median of the variable before
and after the SFN administration for each protocol arm is
calculated. Bar plots and contingency tables are used to describe
the distribution of each ordinal variable. The Wilcoxon signed-rank
test is used to compare the pre- and post- SFN administration
values of each variable. A generalized linear mixed effect model
with random intercept, which accounts for the clustering effect at
the subject level, is used to explore the effects of the SFN
administration and the SFN dose level on each outcome of
interest.
[0200] A subset of the subjects have up to 3 nevi followed
observationally for an extended period of time. Trajectory plots
are used to evaluate the morphological changes over time for these
nevi. A paired t-test is used to test the SFN administration effect
at each time point. A linear mixed effect model is used to evaluate
the SFN administration, SFN dose, and time effect on the
morphological changes of the nevi.
Sample Size Justification
[0201] Table 2 provides the 80% exact confidence intervals for SAE
(small area estimate) rate for different possible outcomes of each
arm.
TABLE-US-00002 TABLE 2 90% Confidence interval (CI) estimates for
SAE rate Number of SAEs observed SAE rate estimate 80% exact CI 1/6
17% (2%, 51%) 2/6 33% (9%, 67%) 3/6 50% (20%, 80%)
[0202] The analysis unit for this protocol is the nevus. Each
subject contributes from 2 to 3 nevi to the overall analysis.
Therefore, the study has a minimum of 6 nevi at the baseline and
day 28 time points for each arm of the study. With 6 nevi, the mean
of each outcome is estimated with an 80% CI width equal to 1.2
times the standard deviation of the corresponding outcome.
[0203] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only examples of
the invention and should not be taken as limiting the scope of the
invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
* * * * *