U.S. patent application number 14/593517 was filed with the patent office on 2015-07-02 for local treatment of neurofibromas.
The applicant listed for this patent is OncoSynergy, Inc.. Invention is credited to Ruihong CHEN, Allan E. RUBENSTEIN, Xiaodong SHEN, Scott STEWART, Jin-Chen YU.
Application Number | 20150182452 14/593517 |
Document ID | / |
Family ID | 36777885 |
Filed Date | 2015-07-02 |
United States Patent
Application |
20150182452 |
Kind Code |
A1 |
CHEN; Ruihong ; et
al. |
July 2, 2015 |
LOCAL TREATMENT OF NEUROFIBROMAS
Abstract
A method for treating a neurofibroma, e.g. dermal neurofibroma,
a subdermal neurofibroma, or a superficial plexiform neurofibroma,
in a subject in need of such treatment is disclosed. The method
comprises locally applying a composition to a neurofibroma either
topically or intralesionally. This method does not encompass
systemic administration of the composition to the subject to have
an effect on the neurofibromas. Compositions useful for such
treatments and methods of preparing the compositions are
disclosed.
Inventors: |
CHEN; Ruihong; (Foster City,
CA) ; RUBENSTEIN; Allan E.; (New York, NY) ;
SHEN; Xiaodong; (Foster City, CA) ; STEWART;
Scott; (San Diego, CA) ; YU; Jin-Chen; (Palo
Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OncoSynergy, Inc. |
San Francisco |
CA |
US |
|
|
Family ID: |
36777885 |
Appl. No.: |
14/593517 |
Filed: |
January 9, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13918223 |
Jun 14, 2013 |
|
|
|
14593517 |
|
|
|
|
13524590 |
Jun 15, 2012 |
8487004 |
|
|
13918223 |
|
|
|
|
11815443 |
Jul 22, 2008 |
8211875 |
|
|
PCT/US2006/003588 |
Feb 2, 2006 |
|
|
|
13524590 |
|
|
|
|
60649854 |
Feb 2, 2005 |
|
|
|
60669813 |
Apr 7, 2005 |
|
|
|
Current U.S.
Class: |
514/43 ; 514/183;
514/274; 514/395; 514/450; 514/463; 514/492; 514/557; 514/567;
514/613; 514/619; 514/77; 514/79 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 37/02 20180101; A61K 31/513 20130101; A61K 31/282 20130101;
A61K 9/0014 20130101; A61K 31/675 20130101; A61P 29/00 20180101;
A61K 31/165 20130101; A61P 35/00 20180101; A61P 25/00 20180101;
A61K 31/7064 20130101; A61K 31/65 20130101; A61K 31/19 20130101;
A61K 31/366 20130101; A61K 31/196 20130101; A61P 37/04 20180101;
A61K 31/167 20130101; A61K 31/395 20130101; A61K 9/0019 20130101;
A61K 31/661 20130101; A61P 43/00 20180101; A61K 31/4745 20130101;
A61K 31/4184 20130101 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 31/282 20060101 A61K031/282; A61K 31/513 20060101
A61K031/513; A61K 31/7064 20060101 A61K031/7064; A61K 31/366
20060101 A61K031/366; A61K 31/395 20060101 A61K031/395; A61K 31/196
20060101 A61K031/196; A61K 31/165 20060101 A61K031/165; A61K 31/19
20060101 A61K031/19; A61K 31/167 20060101 A61K031/167; A61K 31/661
20060101 A61K031/661; A61K 31/675 20060101 A61K031/675; A61K
31/4184 20060101 A61K031/4184 |
Claims
1. A method of treating a dermal neurofibroma, a subdermal
neurofibroma, or a superficial plexiform neurofibroma in a subject
in need of such treatment, which method comprises locally
administering a composition to the region of the tumor, wherein the
composition comprises (a) at least one agent for abating the
negative impact of the neurofibroma on the subject, and optionally
(b) a pharmaceutically acceptable excipient that aids in
transporting the agent into the tumor where it is preferably
maintained for a sufficient period of time to negatively impact the
neurofibroma.
2. The method of claim 1, wherein said local administration is
topically applying said composition to the surface of the skin in
the region of the tumor, and wherein said excipient aids in
transporting the agent across the skin and to the tumor and
preferably maintains the agent on the skin of the subject.
3. The method of claim 1, wherein said local administration is
intralesionally injecting said composition into the tumor.
4. The method of claim 1, wherein the agent comprises a
chemotherapeutic agent, wherein (1) the agent interferes with DNA
replication by topoisomerase inhibition; (2) the agent disrupts the
microtubule and/or mitotic spindle; (3) the agent acts as an
alkylating or damaging agent to DNA; (4) the agent interferes with
nucleotide synthesis or a combination thereof.
5. The method of claim 1, wherein the agent comprises a sclerosing
agent.
6. The method of claim 1, wherein the agent is an immunomodulator,
an immunoregulator, an immunosuppressant, an immunostimulant, or an
non-steroidal anti-inflammatory agent.
7. The method of claim 1, wherein the agent modulates gene
transcription.
8. The method of claim 1, wherein the agent is an angiogenesis
inhibitor.
9. The method of claim 1, wherein the agent alters the structure,
function, localization, or post-translational modification of small
GTPases.
10. The method of claim 1, wherein the agent is a chemopreventative
agent.
11-19. (canceled)
20. A composition useful for treating a dermal neurofribroma, a
subdermal neurofibroma, or a superficial plexiform neurofibroma in
a subject, which composition comprises (a) at least one agent for
abating the negative impact of the neurofribroma on the subject,
and optionally (b) a pharmaceutically acceptable topical excipient
that aids in transporting the agent across the skin into the tumor
and preferably maintains the agent on the subject's skin for a
period of time.
21. The composition of claim 20, wherein the agent is a
chemotherapeutic agent, wherein (1) the agent interferes with DNA
replication by topoisomerase inhibition; (2) the agent disrupts the
microtubule and/or mitotic spindle; (3) the agent acts as an
alkylating or damaging agent to DNA; (4) the agent interferes with
nucleotide synthesis or a combination thereof.
22. The composition of claim 20, wherein the agent comprises a
sclerosing agent.
23. The composition of claim 20, wherein the agent is an
immunomodulator, an immunoregulator, an immunosuppressant, an
immunostimulant, or an non-steroidal anti-inflammatory agent.
24. The composition of claim 20, wherein the agent modulates gene
transcription.
25. The composition of claim 20, wherein the agent is an
angiogenesis inhibitor.
26. The composition of claim 20, wherein the agent alters the
structure, function, localization, or post-translational
modification of small GTPases.
27. The composition of claim 20, wherein the agent is a
chemopreventative agent.
28. The composition of claim 20, wherein the agent is an inhibitor
selected from the group consisting of a protein kinase inhibitor, a
lipid kinase inhibitor, a heat shock protein inhibitor, a protein
chaperone inhibitor, a phosphatase inhibitor or a protease
inhibitor.
29-37. (canceled)
38. A method of preparing a medicament for topically treating a
dermal neurofibroma, a subdermal neurofibroma, or a superficial
plexiform neurofibroma, which method comprises combining at least
one agent for abating the negative impact of a neurofibroma on a
subject with a pharmaceutically acceptable topical excipient that
aids in transporting the agent(s) across the skin into the tumor
and maintains the agent(s) on the skin of the subject for a period
of time.
Description
BACKGROUND OF THE INVENTION
[0001] Neurofibromatosis type 1 (NF1) is the most common single
gene disorder to affect the human nervous system. It is transmitted
genetically in an autosomal dominant manner. NF1 affects
approximately 1.5 million people worldwide and there is no racial,
ethnic, or geographic predilection for the disorder. NF1 is caused
by mutation in the NF1 gene, which produces neurofibromin, a tumor
suppressor. A high spontaneous mutation rate (50%) at the NF1 locus
insure that the disorder is unlikely to decrease significantly in
the population due to genetic screening.
[0002] People affected by NF1 are at increased risk for developing
a variety of tumors of the nervous system, including dermal,
subdermal and plexiform neurofibromas; optic pathway astrocytomas,
and malignant peripheral nerve sheath tumors ("MPNST"), and for
learning disability, scoliosis and certain forms of leukemia. These
tumors may cause disfigurement, nervous system damage and chronic
pain. Dermal neurofibromas are the commonest lesion in NF1 and
occur in 90% of affected individuals. Dermal neurofibromas are
typically small (less than 2 cm in diameter), multiple and first
develop during puberty. They are typically high in collagen
content, have very low metabolic activity, and, as opposed to
plexiform neurofibromas, never undergo malignant degeneration.
There has never been a reported case of malignant degeneration in a
dermal neurofibroma spontaneously or in patients who have received
regional radiotherapy for various malignancies or who have received
chemotherapy for malignancy.
[0003] Certain patients may develop some of the same disfiguring
signs that are associated with Elephant Man's disease, a separate
disorder originally thought to be NF1. The only treatment at the
present time is surgical removal. However, tumors often cannot be
removed without causing major neurologic and/or cosmetic problems,
and frequently re-grow. They are not responsive to radiotherapy or
to known chemotherapeutic agents.
[0004] Cutaneous and subcutaneous neurofibromas may develop at any
time in life, but their numbers are usually small before puberty.
The total number of neurofibromas seen in adults varies from just a
few to thousands. Additional cutaneous and subcutaneous
neurofibromas develop throughout life, although the rate of
appearance may vary greatly from year to year. Other than an actual
cure for the condition, an effective non-surgical treatment for
dermal neurofibromas is the single highest priority for the 100,000
people in the U.S. affected by NFL Dermal neurofibromas cause
significant disfigurement, pain, psychological and financial stress
and should be considered a major unmet medical need in this
population. Development of a local treatment for dermal
neurofibromas has not previously been explored because of several
factors including the relatively low proliferation index with
respect to other tumors, and the fact that the tumors reside
slightly deeper in the skin especially when compared to basal and
squamous cell carcinomas. This local treatment encompasses both
topical treatment and intralesional or intradermal treatment at the
site of the neurofibroma. Therefore, although such a local
treatment of these dermal neurofibromas would be a valuable
treatment for an unmet medical need, work has not been done in this
area previously.
SUMMARY OF THE INVENTION
[0005] One aspect of this invention is a method for treating a
neurofibroma, e.g. dermal neurofibroma, a subdermal neurofibroma,
or a superficial plexiform neurofibroma, in a subject in need of
such treatment. The method comprises locally applying a composition
to a neurofibroma. This method does not encompass systemic
administration of the composition to the subject to have an effect
on the neurofibromas. The method comprises locally administering a
composition to the region of the tumor, wherein the composition
comprises (a) at least one agent for abating the negative impact of
the neurofibroma on the subject, and optionally (b) a
pharmaceutically acceptable excipient that aids in transporting the
agent into the tumor where it is preferably maintained for a
sufficient period of time to negatively impact the
neurofibroma.
[0006] The method more specifically comprises topically applying a
composition to the surface to the skin in the region of the tumor,
wherein the composition comprises (a) at least one agent for
abating the negative impact of a neurofibroma on the subject, and
optionally (b) a pharmaceutically acceptable topical excipient that
aids in transporting the agent across the skin into the tumor and
preferably maintains the agent on the skin of the subject for a
period of time.
[0007] Another aspect of the invention is a composition that is
useful for treating a neurofibroma (e.g. dermal neurofibroma,
subdermal neurofibroma or superficial plexiform neurofibroma) in a
subject. The composition comprises (a) at least one agent for
abating the negative impact of the neurofibroma on the subject, and
optionally (b) a pharmaceutically acceptable topical excipient that
aids in transporting the agent across the skin into the tumor, and
preferably maintains the agent on the subject's skin for a period
of time. The composition alternatively comprises (a) at least one
agent for abating the negative impact of the neurofibroma on the
subject, and optionally, (b) a pharmaceutically acceptable carrier
suitable for injection.
[0008] Still another aspect of the invention is a method of
preparing a medicament for topically treating a neurofibroma (e.g.
dermal neurofibroma, a subdermal neurofibroma, or a superficial
plexiform neurofibroma). The method comprises combining at least
one agent for abating the negative impact on the neurofibroma on a
subject with a pharmaceutically acceptable topical excipient that
aids in transferring the agent across the skin into the tumor and
preferably maintains the agent on the skin of the subject for a
period of time.
[0009] A further aspect of the invention is a method of preparing a
medicament for intralesionally injecting a neurofibroma. The method
comprises combining at least one agent for abating the negative
impact on the neurofibroma on a subject with a pharmaceutically
acceptable excipient that aids in delivering intralesionally into
the tumor so that the agent contacts the tumor of the subject for a
period of time.
[0010] Other aspects of the invention may be apparent to one
skilled in the art after reading the following detailed
description.
DETAILED DESCRIPTION
[0011] In carrying out the method of this invention, a composition
is applied to the surface of the skin in the region of the tumor.
The composition may be applied in any of the known methods for
applying composition. Thus, the composition may be sprayed on,
dabbed on, rubbed on, or adhered onto the skin using a patch or the
like. In addition, it can be applied to the skin and transported
across the skin using a microinjection, electrophoretic,
ultrasound, or radiofrequency mechanism. It can be delivered using
a delivery system that is viral-based or pneumatic. The agent may
be formulated as nanoparticles, dendrimers, or liposomes. The
composition may take a form of a powder, liquid solution or
suspension, a cream, a lotion, an ointment, a gel, or another
composition which allows the composition to be maintained on the
skin for a period of time that is sufficient to cause the agent to
migrate across the skin and to the tumor.
[0012] The composition is applied to the skin in the region of the
tumor or intralesionally injected into the tumor. Generally, the
tumors are readily apparent and will cause disfigurations on the
skin. Thus, the application of the composition will be directly to
the surface of the skin that is in the region of the tumor. That
region will be anywhere on the skin that will be transported across
the skin and into the tumor to cause the active agent in the
composition to act on the tumor. Once the composition is applied to
the skin, it is maintained on the skin for a period of time which
is sufficient to transport the agent across the skin into the tumor
to abate the negative impact of a neurofibroma on the subject.
[0013] For intralesional injection, there are a variety of "active
" drug delivery methods and passive drug delivery (a formulation
without or without a penetration enhancer). All the technologies
perturb the keratin layer of the epidermis to improve drug
delivery, with energy of some type or mechanically. The methods
include: iontophoresis; ultrasound, radiofrequency (RF) and
micro-needles (dermabrasion). Needle-less injection technology,
which is a microneedle attached to a CO2 cartridge which forces
drug through the skin can also be used.
[0014] Abating the negative impact means that (1) the size of the
tumor may stabilize and not increase, (2) the tumor may be reduced
in size, or (3) the pain and/or itching associated with the tumor
may be reduced. Preferably the size of the tumor is reduced
significantly as the disfigurement caused by the neurofibromas is a
major disadvantage of the condition. Markers for determining
whether a composition is acting to abate the negative impact on a
subject include measuring the size of the tumor over a treatment
period and interviewing the subject to determine if there is a
reduction in the level of pain in the subject. Other markers can be
developed such as a proliferation index showing the rate of growth
of the tumor, an apoptotic index showing the rate of death of the
tumor cells, or vessel density. Other biomarkers which may be
apparent to one skilled in the art can also be used to measure the
success of the composition of this invention. For example,
biomarkers that monitor the activation status of Ras-Raf-MEK-MAPK.
pathway may be used to measure target inhibition in tumor tissues
by the composition of this invention. In most cases, a baseline is
established before treatment by measuring the size of the tumor
using a calipers or using a MRI imaging technique, or establishing
an index for the untreated tumor. Once the baseline is established
the treatment can begin and measurements can be taken periodically
to determine the success of the treatment.
[0015] The agents that are useful as a single agent or as a
combination of one or more agents in the composition for applying
to the surface of the skin or intralesionally injected into the
tumor are those that will abate the negative impact of the
neurofibroma on the subject. Preferred agents useful in the present
invention are agents that are not carcinogenic. The FDA or IARC
Monographs have lists of compounds/agents that are considered to be
carcinogenic and not suggest or allowed for human use. Even though,
bleomycin is listed below as an agent, it is not desirable as a
preferred agent because it is carcinogenic. For example, if Such
agents may be a chemotherapeutic agent (e.g., an anti-neoplastic
agent, a cytotoxin, or an anti-proliferative agent); a sclerosing
agent; an immunomodulator (e.g. an immunoregulator, an
immunosuppressant, or an immunostimulant) or an anti-inflammatory
agent, such as a nonsteroidal anti-inflammatory drugs (NSAID) or a
Cox-1 & 2 inhibitor; an agent that modulates gene
transcription, e.g. a HDAC (historic deactylase) inhibitor (e.g.
valproic acid, FK228, trapoxin), an angiogenesis inhibitor; an
agent that alters the structure, function, localization, or
post-translational modification of small GTPases (e.g. famesyl
transferase inhibitors [R115777], isoprenyl cysteine transferase
inhibitors (cysmethnil) an agent that acts as a chemopreventative
agent such as vitamins, vitamin derivatives, antioxidants,
nutritional supplements (e.g. fenretinide, green tea extract
containing EGCG); an antifibrotic agent, an agent that targets
apoptotic or an anti-apoptotic signaling, a kinase inhibitor (e.g.
a protein kinase inhibitor or lipid kinase inhibitor); an
alkylphospholipid; a protein chaperone inhibitor, such as a heat
shock protein inhibitor (e.g., HSP90); an antifungal agent; an
agent that restores the function of a mutated gene, such as an
agent that suppresses non-sense mutations (e.g., gentamicin); a
nucleic acid-based therapeutic agent, a phosphatase inhibitor, a
protease inhibitor an or an agent that inhibits skin hyperplasia
(e.g., actinic keratosis, melanoma, Kaposi's sarcoma, basal cell or
squamous call carcinomas, or skin metastases of other cancers). For
topical treatment, the composition preferably contains a skin
penetrant that aids in pushing the agent across the skin into the
tumor.
[0016] Examples of chemotherapeutic agents that are useful are (1)
agents that interfere with DNA replication by topoisomerase
inhibition, (2) agents that disrupt the microtubule and/or mitotic
spindle, (3) agents that act as alkylating or damaging agents to
DNA, or (4) agents that interfere with nucleotide synthesis, i.e.,
antimetabolites. Specific chemotherapeutic agents include
5-fluorouracil (5-FU) and thiotepa. Each of these materials is well
known by one of ordinary skill in the art and can be obtained from
standard sources.
[0017] Protein kinase inhibitors are compounds that target kinases
that are hyperactivated in neurofibromas, such as components of the
Ras-activated mitogen activated protein kinase pathway (e.g. MEK
and Raf kinases), receptor and non-receptor tyrosine kinases, and
the AKT-mTOR pathway, which promote cell proliferation and
survival, respectively. Lipid kinase inhibitors are compounds that
target the phosphatidylinositol 3-kinase pathway, which is
hyperactivated in neurofibromas and leads to enhanced cell
survival.
[0018] Protein chaperone inhibitors are those that inhibit Heat
Shock Protein or peptidyl-prolyl isomerase function. Heat Shock
Protein activity is required to maintain physiological protein
levels of Raf and KSR, two components of the Ras-Mitogen activated
protein kinase pathway, in addition to numerous other signaling
molecules.
[0019] Angiogenesis inhibitors are those that block (1)
angiogenesis signaling cascade, such as Vascular Endothelial Growth
Factor (VEGF) Receptor signaling, (2) extracellular matrix
breakdown, such as that induced by the matrix metalloproteinases
(e.g., halofuginone), (3) growth, survival, and migration of
endothelial cells, or (4) unknown mechanism of action. Some of
these compounds also fall under other categories. These angiogenic
processes, among others, are needed to ensure adequate blood supply
to a growing tumor and are attractive modes of therapeutic
intervention for NF1.
[0020] Cell-cell communication, both chemical and physical, between
numerous cell types, including Schwann cells, mast cells, and
fibroblasts is likely to be essential for neurofibroma disease
progression. Immunomodulators, such as calcineurin inhibitors that
block transcription of cytokines and growth factors, are an
efficient means to interfere with cell-cell signaling.
[0021] Agents which correct a specific mutation, such as gene
replacement or gentamicin for repair of nonsense mutations, are
also useful,
[0022] The present invention is more specifically described by the
following preferred embodiments:
[0023] 1. A method of treating a dermal neurofibroma, a subdermal
neurofibroma, or a superficial plexiform neurofibroma in a subject
in need of such treatment, which method comprises locally
administering a composition to the region of the tumor, wherein the
composition comprises (a) at least one agent for abating the
negative impact of the neurofibroma on the subject, and optionally
(b) a pharmaceutically acceptable excipient that aids in
transporting the agent into the tumor where it is preferably
maintained for a sufficient period of time to negatively impact the
neurofibroma. 2. The method of 1, wherein said local administration
is topically applying said composition to the surface of the skin
in the region of the tumor, and wherein said excipient aids in
transporting the agent across the skin and to the tumor and
preferably maintains the agent on the skin of the subject. 3. The
method of 1, wherein said local administration is intralesionally
injecting said composition into the tumor. 4. The method of any one
of 1-3, wherein the agent comprises a chemotherapeutic agent,
wherein (1) the agent interferes with DNA replication by
topoisomerase inhibition; (2) the agent disrupts the microtubule
and/or mitotic spindle; (3) the agent acts as an alkylating or
damaging agent to DNA; (4) the agent interferes with nucleotide
synthesis or a combination thereof. 5. The method of any one of
1-3, wherein the agent comprises a sclerosing agent. 6. The method
of any one of 1-3, wherein the agent is an immunomodulator, an
immunoregulator, an immunosuppressant, an immunostimulant, or an
non-steroidal anti-inflammatory agent. 7. The method of any one of
1-3, wherein the agent modulates gene transcription. The method of
any one of 1-3, wherein the agent is an angiogenesis inhibitor. 9.
The method of any one of 1-3, wherein the agent alters the
structure, function, localization, or post-translational
modification of small GTPases. 10. The method of any one of 1-3,
wherein the agent is a chemopreventative agent. 11. The method of
any one of 1-3, wherein the agent is an inhibitor selected from the
group consisting of a protein kinase inhibitor, a lipid kinase
inhibitor, a heat shock protein inhibitor, a protein chaperone
inhibitor, a phosphatase inhibitor or a protease inhibitor. 12. The
method of any one of 1-3, wherein the agent is an anti-fibrotic
agent. 13. The method of any one of 1-3, wherein the agent is an
alkylphospholipid. 14. The method of any one of 1-3, wherein the
agent targets apoptotic or anti-apopotic signaling. 15. The method
of any one of 1-3, wherein the agent is a nucleic acid-based
therapeutic agent. 16. The method of any one of 1-3, where the
agent restores the function of a mutated gene. 17. The method of
any one of 1-3, wherein the agent inhibits skin hyperplasia. 18.
The method of any one of 1-3, wherein the agent is an alkylating
agent, such as thiotepa or carboplatin; an anti-metabolite or a
nucleoside analogue, such as 5-fluorouracil, triciribine,
sangivamycin, or tubercidin, a topoisomerase inhibitor, such as
podophyllotoxin, a microtubule inhibitor, such as mebendazole, a
sclerosing agent, such as bleomycin, doxycycline or analogues
thereof an anti-inflammatory agent or a nonsteroidal
anti-inflammatory agent (NSAID), such as diclofenac; an agent that
modulates gene transcription, such as a HDAC inhibitor comprising
tricostatin A or valproic acid; a chemopreventative agent, such as
a retinoid, such as fenretinide; an alkylphospholipid, such as
miltefosine; a HSP90 inhibitor, such as geldanamycin derivatives,
such as 17-AAG, radicicol or analogues thereof; halofuginone,
gentamicin, rapamycin, or a combination thereof. 19. The method of
1-18, wherein the excipient includes a skin penetrant.
[0024] Also disclosed are preferred compositions useful for
treatment of neurofibromas. 20. A composition useful for treating a
dermal neurofribroma, a subdermal neurofibroma, or a superficial
plexiform neurofibroma in a subject, which composition comprises
(a) at least one agent for abating the negative impact of the
neurofribroma on the subject, and optionally (b) a pharmaceutically
acceptable topical excipient that aids in transporting the agent
across the skin into the tumor and preferably maintains the agent
on the subject's skin for a period of time. 21. The composition of
20, wherein the agent is a chemotherapeutic agent, wherein (1) the
agent interferes with DNA replication by topoisomerase inhibition;
(2) the agent disrupts the microtubule and/or mitotic spindle; (3)
the agent acts as an alkylating or damaging agent to DNA; (4) the
agent interferes with nucleotide synthesis or a combination
thereof. 22. The composition of 20, wherein the agent comprises a
sclerosing agent. 23. The composition of 20, wherein the agent is
an immunomodulator, an immunoregulator, an immunosuppressant, an
immunostimulant, or an non-steroidal anti-inflammatory agent. 24.
The composition of 20, wherein the agent modulates gene
transcription. 25. The composition of 20, wherein the agent is an
angio genesis inhibitor. 26. The composition of 20, wherein the
agent alters the structure, function, localization, or
post-translational modification of small GTPases. 27. The
composition of 20, wherein the agent is a chemopreventative agent.
28 The composition of 20, wherein the agent is an inhibitor
selected from the group consisting of a protein kinase inhibitor, a
lipid kinase inhibitor, a heat shock protein inhibitor, a protein
chaperone inhibitor, a phosphatase inhibitor or a protease
inhibitor. 29. The composition of 20, wherein the agent is an
anti-fibrotic agent. 30. The composition of 20, wherein the agent
is an alkylphospholipid. 31. The composition of 20, wherein the
agent targets apoptotic or anti-apopotic signaling. 32. The
composition of 20, wherein the agent is a nucleic acid-based
therapeutic agent. 33. The composition of 20, where the agent
restores the function of a mutated gene. 34. The composition of 20,
wherein the agent inhibits skin hyperplasia. 35. The composition of
20, wherein the agent is an alkylating agent, such as thiotepa or
carboplatin; an anti-metabolite or a nucleoside analogue, such as
5-fluorouracil, triciribine, sangivamycin, or tubercidin, a
topoisomerase inhibitor, such as podophyllotoxin, a microtubule
inhibitor, such as mebendazole, a sclerosing agent, such as
bleomycin, doxycycline or analogues thereof; an anti-inflammatory
agent or a nonsteroidal anti-inflammatory agent (NSAID), such as
diclofenac; an agent that modulates gene transcription, such as a
HDAC inhibitor comprising tricostatin A or valproic acid; a
chemopreventative agent, such as a retinoid, such as fenretinide;
an alkylphospholipid, such as miltefosine; a HSP90 inhibitor, such
as geldanamycin derivatives, such as 17-AAG, radicicol or analogues
thereof; halofuginone, gentamicin, rapamycin, or a combination
thereof. 36. The composition of 20-36, wherein the excipient
includes a skin penetrant. 37. Use of the composition of any one of
20-36 for the preparation of a pharmaceutical composition for the
treatment of neurofibromas in a subject. 38. A method of preparing
a medicament comprising the composition of any one of 20-36 for
topically treating a dermal neurofibroma, a subdermal neurofibroma,
or a superficial plexiform neurofibroma, which method comprises
combining at least one agent for abating the negative impact of the
neurofibroma on the subject with a pharmaceutically acceptable
topical excipient that aids in transporting the agent(s) across the
skin into the tumor and preferably maintains the agent(s) on the
skin of the subject for a period of time.
[0025] Examples of specific compounds alone or in combination that
are useful in this invention as an agent for abating the negative
impact of a neurofibroma on a subject include thiotepa,
doxycycline, bleomycin, diclofenac, carboplatin, 5-fluorouracil
(5-FU), mebendazole, halfuginone, gentamycin, rapamycin,
miltefosine, and the like.
[0026] Further, the agent(s) contained in the compositions either
as the single pharmaceutically active agent or in combination with
one or more pharmaceutically active agents with or without a
pharmaceutically acceptable topical excipient for abating the
negative impact of the neurofibroma on a subject, includes but are
not limited to the following agents: [0027] 1. Chemotherapeutic
agents, such as cytotoxins, anti-neoplastic or anti-proliferative
agents, for example: agents such as: [0028] An alkylating agent,
such as Thiotepa,
[0028] ##STR00001## [0029] A topoisomerase inhibitor, such as
Camptothecin,
[0029] ##STR00002## [0030] Carboplatin,
[0030] ##STR00003## [0031] An antimetabolite, such as
5-fluorouracil (5-FU)
[0031] ##STR00004## [0032] A topoisomerase inhibitor, such as
Podophyllotoxin,
[0032] ##STR00005## [0033] A microtubule inhibitor, such as
Mebendazole,
[0033] ##STR00006## [0034] An antimetabolite or a nucleoside
analogue, such as Cladribine
[0034] ##STR00007## [0035] A topoisomerase inhibitor, such as XK469
(2-(4-((7-Chloro-2-quinoxalinyl)oxy)phenoxy)propionic acid),
[0035] ##STR00008## [0036] A nucleoside analogue, such as
Sangivamycin
[0036] ##STR00009## [0037] A nucleoside analogue, such as
Tubercidin,
[0037] ##STR00010## [0038] 2. Sclerosing agents, for example:
[0039] Bleomycin, an antineoplastic antibiotic,
[0039] ##STR00011## [0040] A tetracycline analogue or antibiotic,
such as Doxycycline
[0040] ##STR00012## [0041] 3. Immunomodulators or immunoregulators
or immuno stimulants or immunosuppressants, for example: [0042]
Immunosimulants, such as Imiquimod (immune response modifier or
TLR7/8 ligand or agonist),
[0042] ##STR00013## [0043] Immunosuppressants, such as Tacrolimus
(FK506)
[0043] ##STR00014## [0044] 4. An anti-inflammatory agent or
non-steroidal anti-inflammatory drug (NSAID), such as: [0045]
Diclofenac
[0045] ##STR00015## [0046] Celecoxib (also a Cox-1 & 2
inhibitor or an anti-inflammatory agent),
[0046] ##STR00016## [0047] 5. Agents that modulate gene
transcription e.g. HDAC (histone deactylase) inhibitors, for
example: [0048] A fatty acid class if HDAC inhibitors, such as
Valproic acid,
[0048] ##STR00017## [0049] A hydroxamic acid class of HDAC
inhibitors, such as Tricostatin A (TSA)
[0049] ##STR00018## [0050] Carbamazepine (below) and its
derivatives such as carbamazepine epoxide,
[0050] ##STR00019## [0051] A cyclic tetrapeptide class of HDAC
inhibitors, such as depsipeptide FK228 (FR901228),
[0051] ##STR00020## [0052] 6. Angiogenesis inhibitors, for example:
[0053] A fumagillin, which is secreted by the fungus Aspergillus
fumigatus, and its analogues such as TNP-470
[O-(Chloroacetylcarbamoyl) fumagillol] shown below:
[0053] ##STR00021## [0054] Thalidomide, also an immunomodulatory
agent
[0054] ##STR00022## [0055] 7. Agents that inhibit the cellular
process that required for the modification of small GTPases such
as: [0056] Farnesyl transferase inhibitors [e.g. R115777,
(B)-6-[amino(4-chlorophenyl)(1-memethyl-1H-imidazol-5yl)methyli4-(3
chiorophenyl)-1-methyl-2(1H)-quinolinone],
[0056] ##STR00023## [0057] Isoprenyl cysteine transferase
inhibitors [e.g. cysmethnil]
[0057] ##STR00024## [0058] HMG-CoA inhibitors (statins, e.g.
lovastatin)
[0058] ##STR00025## [0059] Bisphosphonates (e.g. alendronate, as
inhibitors of mevalonate pathway)
[0059] ##STR00026## [0060] 8. Chemopreventative agents, for
example: [0061] Synthetic retinoids (Fenretinide)
[0061] ##STR00027## [0062] Retinoic acid
[3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexenyl)-nona-2,4,6,8-tetraenoic
acid] and analogs,
[0062] ##STR00028## [0063] Curcumin and derivatives,
[0063] ##STR00029## [0064] EGCG, (-)-Epigallocatechin Gallate (see
below) and analogues
[0064] ##STR00030## [0065] I3C (Indole-3-carbinol),
[0065] ##STR00031## [0066] Cyclopamine,
[0066] ##STR00032## [0067] Methylglyoxal
[0067] ##STR00033## [0068] 9. Kinase inhibitors, MEK inhibitors,
Raf inhibitors, mTOR inhibitors, PI3K inhibitors, examples, such
as: [0069] MEK inhibitors, such as U0126
[1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene],
[0069] ##STR00034## [0070] P13K inhibitors, such as Ly294002
[2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]
[0070] ##STR00035## [0071] EGFR/ErbB2 inhibitors, such as
4-(4-Benzyloxyanilino)-6,7-dimethoxyquinazoline,
[0071] ##STR00036## [0072] AG1478 (EGFR inhibitor),
4-(3-Chloroanillino)-6,7-dimethoxyquinazoline
[0072] ##STR00037## [0073] Inhibitors of multiple Tyrosine kinases,
e.g. PDGFR inhibitors such as KN2941
(4-(6.7-dimethoxy-4-quinazolinyl)-N-(3,4-methylenedioxybenzyl)-1-piperazi-
nethiocarboxamide)
[0073] ##STR00038## [0074] Inhibitors of multiple kinases such as
SU11652,
5[(Z)-(5-Chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[-
2(diethylamino)ethyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide
[0074] ##STR00039## [0075] Inhibitors of multiple kinases such as
SU11248 (Trade name is Sutent),
5-[(Z)-(5-Fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-(dieth-
ylamino)ethyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide
[0075] ##STR00040## [0076] Inhibitors of multiple kinases e.g.
VEGFR2 and Raf kinases such as Bay43-9006 (NEXAVAR, or Sorafenib),
4-(4-{3-4-Chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-N2-methylpyrid-
ine-2-carboxamide
[0076] ##STR00041## [0077] mTOR inhibitors, such as Rapamycin (also
an immunosuppressant, see below) and prodrugs or analogue,
[0077] ##STR00042## [0078] Rho kinase inhibitors such
1-(5-Isoquinolinesulfonyl)homopiperazine]
[0078] ##STR00043## [0079] 10. Novel antifibrotics, which may
include agents that are also angiogenesis inhibitors, for example:
[0080] Halofuginone hydrobromide
[(.+-./-)-trans-7-Bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxoprop-
yl]-4(3H)-quinazolinone hydrobromide]
[0080] ##STR00044## [0081] Pirfenidone
(5-Methyl-N-phenyl-2-1H-pyridone-d5)
[0081] ##STR00045## [0082] 11. Alkylphospholipids, for example:
[0083] A. Miltefosine (HePC) [0084] B. Edelfosine (Et-Et-18-OCH3
[0085] C. Perifosine (D21266)
[0085] ##STR00046## [0086] 12. HSP90 inhibitors (heat shock protein
inhibitors) [0087] Geldanamycin analogues/derivatives such as
17-AAG and its derivatives, 1 7-AAG,
17-7-(Allylamino)-17-demethoxygeldanamycin,
[0087] ##STR00047## [0088] Radicicol and analogues,
[0088] ##STR00048## [0089] Hypericin
[0089] ##STR00049## [0090] 13. Antifungal agents: [0091]
Clotrimazole and analogs,
[0091] ##STR00050## [0092] 14. Agents that restore the function of
a mutated gene, e.g. agents that suppress non-sense mutations, for
example: [0093] Gentamicin
##STR00051##
[0094] In preparing compositions of this invention one is guided by
the teachings of (1) Remingtons Pharmaceutical Sciences, Mack
Publishing Co., 17.sup.th, 18.sup.th, or 19.sup.th Edition,
ISBN:0-912734-04,3, (Also called "Remington: The Science and
Practice of Pharmacy"); (2) Pharmaceutics The Science of Dosage
Form Design, Aulton, Churchill Livingston; (3) Appleton and Lange
Review of Pharmacy (7th 8th Ed) by Hall and Reiss, Appleton and or
Lange; and the like, all of which are incorporated herein by
reference. For nanoparticle or liposome delivery, more information
is available at http://www.happl.com/special/mar013.htm and
http://www.collabo.com/liposomeltm. Information relating to
dendrimer technology is found at
http://www.ringer.com/dedrimer/one.htm.
EXAMPLES
[0095] Agents useful to treat neurofibromas in the present
invention may be selected by several methods described herein. For
example, various compounds/agents described herein as well as other
agents that may be selected are tested in cell-based assay for
their potency to inhibit cell proliferation of a panel of NE1
related cell lines, are tested to determine an effect on dermal
neurofibroma explant assays, and are tested to determine an effect
on xenograft models for dermal neurofibromas.
I. In Vitro Assays
1) Cell Proliferation Assays
[0096] Several physiologically relevant cells or cell lines were
employed to measure compound potency in cell proliferation assays.
These cells include: (1) NEI-deficient human malignant peripheral
nerve sheath tumor (MPNST) cells, (2) Nf.sup.+.about./.about. mouse
embryonic Schwann cells, and (3) Nf1- and p53-deficient mouse cell.
Mouse Schwann cells are grown on plates coated with larninin and
cultured in Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F12
medium (F12), a 1:1 blend of DMEM and F12 media, supplemented with
recombinant heregulin-.beta.1 (10 ng/ml), forskolin (2.mu., M), and
N2 supplement (Invitrogen, California. Cat#17502-048). All the
other cells are grown in DMEM with 10% fetal bovine serum (FBS).
The following publications disclose the preparation of the cell
lines used in the cell-proliferation assay. [0097] Basu, T. N.,
Gutmann, D. H., Fletcher, J. A., Glover, T. W., Collins, F. S., and
Downward, J. (1992) Nature 356, 713-715 [0098] DeClue, J. E.,
Papageorge, A. G., Fletcher, J. A., Diehl, S. R., Ratner, N., Vass,
W. C., and Lowy, D. R. (1992) Cell 69, 265-273 [0099] DeClue, J.
E., Heffelfnger, S., Benvenuto, G., Ling, B., Li, S., Rui, W.,
Vass, W. C., Viskochil, D., and Ratner, N. (2000) J Clin Invest
105, 1233-1241 [0100] Vogel, K. S., Klesse, L. J., Velasco-Miguel,
S., Meyers, K., Rushing, E. J., and Parada, L. F. (1999) Science
286, 2176-2179 [0101] Manent, J., Oguievetskaia, K., Bayer, J.,
Ratner, N., and Giovannini, M. (2003) J Neurosci Methods 123,
167-173 [0102] Morrissey, T. K., Kleitman, N., and Bunge, R. P.
(1991) J Neurosci 11, 2433-2442 [0103] Verdu, B., Rodriguez, F. S.,
Gudino-Cabrera, G., Nieto-Sampedro, M., and Navarro, X. (2000) J
Neurosci Methods 99, 111-117 [0104] Kim, H. A., Ling, B., and
Ratner, N. (1997) Mol Cell Biol 17, 862-872 [0105] Zhu, Y., Ghosh,
P., Charnay, P., Burns, D. K., and Parada, L. F. (2002) Science
296, 920-922 [0106] Zhu, Y., Romero, M. I., Ghosh, P., Ye, Z.,
Charnay, P., Rushing, E. J., Marth, J. D., and Parada, L. F. (2001)
Genes Dev 15, 859-876 [0107] Muir, D., Neubauer, D., Lim, I. T.,
Yachnis, A. T., and Wallace, M. R. (2001) Am J Pathol 158, 501-513
[0108] Li, Y., Rao, P. K., Wen, R., Song, Y., Muir, D., Wallace,
P., van Home, S. J., Tennekoon, G. I., and Kadesch, T. (2004)
Oncogene 23, 1146-1152
[0109] To perform cell proliferation assays, appropriate number of
cells that can reach -70% confluence in 3 days (for example,
3000-6000 cell/well for human MPNST cells and mouse
Nf1/p53-deficient cells, 8000-10000 cell/well for mouse embryonic
Schwann cell) were plated in 96-well plates. Various concentrations
of each compound/agent to be tested were added to the growing media
and the cells were then cultured for 3 days. Upon completion of the
incubation, media were gently removed and 100 pi of ATPlite
solution (Perkin Elmer, Boston, Cat.# 6016941) was added in each
well. Viable cells were measured by detecting luminescence
generated from reaction of ATPlite solution and the ATF in the
cells. Potency ranges of various compounds/agents to inhibit cell
proliferation of several NM-related cell lines were determined, The
selection criteria for agents/compounds that are useful as good
candidates for the local treatment of neurofibromas is based on the
selection of any compounds with an IC5Os equal or below 10 .mu.M
(IC50 is defined as the concentration of an inhibitor that is
required for inhibition of cell proliferation by 50%.) Compounds
that meet such criteria are: alkylating agents (e.g. Thiotepa),
anti-metabolites or nucleoside analogues (e.g. 5-fluorouracil,
Sangivamycin, Tubercidin, Triciribine, Cladribine), topoisomerase
inhibitors (e.g. Camptothecin, Podophyllotoxin, XK469), microtubule
inhibitors (e.g. Mebendazole), sclerosing agents. (e.g. Bleomycin,
Doxycyleline and analogues), agents that modulate gene
transcription (HDAC inhibitors, such as Tricostatin A),
chemopreventative agents (e.g. retinoids such as Fenretinide,
retinoic acid and analogues, curcumin and derivatives, EGCG and
analogues, and methylglyoxal), kinase inhibitors (e.g. U0126,
LY294002, EGFR/ErbB2 inhibitors, KN2941, SU11652, Bay43-9006,
rapamycin and their analogues), alkylphospholipids (e.g.
miltefosine), HSP90 inhibitors (e.g. Geldanamycin derivatives such
as 17-AAG, radicicol and analogues, hypericin), anti-fungal agents
(e.g. clotrimazole), agents that suppress non-sense mutations (e.g.
gentamicin). In addition, if compounds are believed to have
mechanisms of actions that are non-cell autonomous (e.g.
immunomodulatory or anti-angiogenesis) or unknown, they may not
score as positives but they will be tested in explant models,
xenograft models, and in the proof-of-principle clinical trials
with NF1 patients if possible.
2) Explant Assay
[0110] The efficacy of the compound/agent was tested in an ex vivo
tumor explant model as follows. Fresh human dermal neurofibroma was
cut into eight small pieces and cultured on standard cell culture
plates with medium containing DMEM/F12, 10% FBS, heregulin and
forskolin. The pieces were divided into treatment and control
groups. Compound at different doses was added to the medium on day
1. No compound was added in the negative control group. All tumor
pieces were collected on day 3 and processed for histology by
formalin fixation and paraffin embedding. Tissue sections were cut
and stained with hernatoxylin-eosin for histological analysis. The
histological analysis of the paired explant samples was conducted
blindly. Three histological parameters were used to score the
samples from "-" to "+++": 1) overall tissue integrity; we evaluate
tissue integrity by comparing the cellularity of treated samples to
the negative control samples. The low cellularity area was defined
as >30% less nucleus than the similar area on the control
sample. The photograph of each sample at 40.times. under a bright
field microscopy was take using MagnaFire digital camera
(Optronics, USA) and stored in computer. The low cellularity areas
in the pictures were measured using Photoshop and calculated as
Length.times.Width, and then expressed as a percentage over the
total area of the sample. Overall tissue integrity was scored using
0 to 2+ score. In short, good tissue integrity is scored 0
(<5%), 1+ indicates medium tissue integrity (5-25%), 2+ low
tissue integrity (>25%). Only 2+ cases were regarded as positive
"+" for the final total score; 2) cell death--slides were evaluated
by bright field microscopy. At least 100 cells per view were
counted. Cell death was scored semi-quantitatively using the 0 to
3+ score. The absence of condensed or fragmented nucleus is scored
0, 1+ indicates the lowest level of detectable cell death
(<10%), 2+ moderate cell death (10-20%), and 3+ high cell death
(>30%). All 2+ and 3+ cases were regarded as positive "+" for
the final total score; and 3) effect on vasculatures.--slides were
evaluated by bright field microscopy. The effect on vasculature in
treated samples was determined by comparing with the vasculatures
in control negative samples. At least 20 vasculatures per sample
were counted. Effect on vasculatures was scored semi-quantitatively
using the 0 to 3+ score. In short, absence or the lowest level of
damaged vasculature is scored 0 (<5%), 1+ indicates of some
damaged vasculature (5-25.degree. A), 2+ high (>25%). All 1+ and
2-i-cases were regarded as positive "+" for the final total score.
Final histological score was the sum of the scores of the above
three parameters, expressed as "-" to "+++". Any compound with a
histological score of "+" or more will be potential good candidates
for local treatment of nenrofibromas. Compounds that meet such
criteria are: alkylating agents (e.g. Thiotepa, Carboplatin),
anti-metabolites or nucleoside analogues (e.g. 5-fluorouracil,
Sangivamycin, Tubercidin), topoisomerase inhibitors (e.g.
Podophyllotoxin), microtubule inhibitors (e.g. Mebendazole),
sclerosing ;agents (e.g. Bleomycin), anti-inflammatory agents or
nonsteroidal anti-inflammatory agents (NSAIDs) (e.g. Diclofenac),
agents that modulate gene transcription (HDAC inhibitors, such as
Tricostatin A, Valproic acid), chemopreventative agents (e.g.
retinoids such as Fenretinide), HSP90 inhibitors (e.g. Geldanamycin
derivatives such as 17-AAG, radicicol and analogues).
3) Xenograft Assay
[0111] The efficacy of the compound was tested in a tumor burden
reduction study in a mouse dermal neurofiberoma (DNF) xenograft
model as follows. Female SCID mice between 5 and. 6 weeks of age
and weighing approximately 20 g were obtained from Charles River
Laboratories (Wilmington, Mass.). Animals were implanted
subcutaneously with fragments of fresh human DNF. 3 days following
inoculation, the animals were randomly allocated into treatment and
control groups. Each group contained 6 tumored mice, Each mouse was
ear-tagged and followed individually throughout the experiment.
Dosing started on Day 1 following randomization. The compound was
administered intralesionally on a twice weekly schedule for 4
weeks. Compound plus vehicle was administered at different doses in
the volume of 50 Vehicle alone was administered to serve as the
negative control, Mice were weighed twice weekly, and tumor
measurements were obtained using calipers twice weekly, starting on
Day 1. Tumor volumes were calculated by the standard formula
(W.sup.2.times.L)/2, where L is the length and W is the width.
(Blaskovich M A, Lin Q, Delarue F L, Sun 5, Park H S, Coppola D,
Hamilton A D, Sebti S M (2000), Design of GFB-111, a
platelet-derived growth factor binding molecule with antiangiogenic
and anticancer activity against human tumors in mice. Nat
Biotechnol 18: 1065-1070). The tumor fragments were weighed before
inoculation and after sacrifice. The differences were expressed as
the percentage of original tumor weight. The harvested tumors were
processed for histology by formalin fixation and paraffin
embedding. Tissue sections were cut and stained with
hematoxylin-eosin for histological analysis. The effect of the
compound on xenografts was determined by either of: 1) tumor weight
measurements--the tumor weight differences before and after
treatment in both treated and control groups were analyzed using
Student's t test. The result of a certain dose of the compound is
considered to be "+" when the difference in tumor weight is
statistically significant (p<0.05). 2) histological
analysis--three histological parameters were used to score the
samples. A) tissue necrosis--the picture of each sample at
40.times. under a bright field microscopy was take using MagnaFire
digital camera (Optronics, USA) and stored in computer. The
necrosis areas in the pictures were measured using Photoshop and
calculated as Leath.times.Width, and then expressed as a percentage
over the total area of the sample. Overall tissue necrosis was
scored. using 0 to 2+ score. In short, absence of necrosis is
scored 0 (<5%), 1+ indicates medium tissue necrosis (5-25%), 2+
high tissue necrosis (>25%). Only 2+ cases were regarded as
positive "+" for the final total score. B) tissue cellularity--the
low cellularity area in the treated samples was defined as >30%
less nucleus than the similar area on the control sample. The
picture of each sample at 40.times. under a bright field microscopy
was take using MagnaFire digital camera (Optronics, USA) and stored
in computer. The low cellularity areas in the pictures were
measured using Photoshop and calculated as Length.times.Width, and
then expressed as a percentage over the total area of the sample.
Overall tissue integrity was scored using 0 to 2+ score. In short,
good tissue integrity is scored 0 (<5%), 1+ indicates medium
tissue integrity (5-25%), 2+ low tissue integrity (>25%). All 1+
and 2+ cases were regarded as positive "+" for the final total
score. C) inflammatory cells infiltration--slides were evaluated by
bright field microscopy. At least 100 cells per view were counted
and the number of inflammatory cells per 100 total cells was
calculated as a percentage for each sample. Inflammatory cells
infiltration was determined by the difference between the
percentage of the treated samples with that of the control sample
and scored semi-quantitatively using the 0 to 3+ score. In short,
absence of or very low difference in inflammatory cells
infiltration is scored 0 (<5%), 1+ indicates medium level of
Inflammatory cells infiltration (5-25%), and 2+ high level of
inflammatory cells infiltration (>25%). All 1+ and 2+ cases were
regarded as positive "+" for the final total score. Final
histological score was the sum of the scores of the above three
parameters, expressed as "-" to "+++". Any compound with either a
"+" in tumor weight measurement or a histological score of "+" or
more will be potential good candidates for local treatment of
neurofibromas. Compounds that meet such criteria are: alkylating
agents (e.g. Thiotepa, Carboplatin), anti-metabolites or nucleoside
analogues (e.g. 5-fluorouracil, Sangivamycin, Tubercidin),
topoisomerase inhibitors (e.g. Podophyllotoxin), microtubule
inhibitors (e.g. Mebendazole), .sclerosing agents. (e.g.
131.0Ornyeiti;i:PoxycyOline); anti-inflammatory agents or
nonsteroidal anti-inflammatory agents (NSAID) (e.g. Diclofenac),
agents that modulate gene transcription (HDAC inhibitors, such as
Tricostatin A, Valproic acid), chemopreventative agents (e.g.
retinoids such as Fenretinide), alkylphospholipids (e.g.
miltefosine), HSP90 inhibitors (e.g. Geldanamycin derivatives such
as 17-AAG, radicicol and analogues).
[0112] The compounds/agents above were tested using one or more of
the above assays to select agents useful in the present invention
to treat neurofibromas: The assays are preferably run in a
particular order and when "positive results" are obtained, then the
next assay is performed and so on. For example if an agent is
tested in the cell proliferation assay and is positive by the
criteria disclosed above, then this agent is tested in the explant
assay, and if positive according to the above criteria, then this
agent is tested in the xenograft assay. If all three of the assays
results in positive results based on the criteria for a specific
agent or compound, then this agent expected to be a good candidate
for successful treatment of neurofibromas, specifically NF1.). In
addition, for compounds that arc believed to have mechanisms of
actions that are non-cell autonomous (e.g. immunomodulatory or
anti-angiogenesis) or unknown, they are expected to be good
candidates for successful treatment of neurofibromas if positive
results are obtained in xenograft models, or in the
proof-of-principle clinical trials with NF1 patients if
possible.
II. In Vivo Studies
General Stud Design for Local Treatment of Neurofibromas with an
Agent or Compound
A. Intralesional Administration
[0113] The study was a proof-of-concept, prospective, safety and
efficacy study of the effect of intralesionally administered drugs
given once a week to 3 target cutaneous neurofibromas for 4
consecutive weeks. Six subjects were enrolled. All subjects had the
diagnosis of NF1 and possessed at least 6 cutaneous neurofibromas
0.5 to 1.5 cm, inclusive, on the back. All subjects were from 18 to
65 years of age, inclusive. All subjects otherwise were in good
health, or had stable concomitant medical conditions appropriately
managed by a primary care physician. Enrolled subjects demonstrated
no clinically significant abnormalities on both laboratory and
physical examination, other than the features of NF1. After
screening and enrollment, all subjects received a defined volume
(based on the size of the lesion) of intralesional drug once weekly
into 3 target cutaneous neurofibromas that are 0.5 to 1.5 cm in
diameter, inclusive, in its largest dimension. Each target
cutaneous neurofibroma was different in size, with one 0.5 to 0.8
cm in diameter, a second between 0.81 to 1.2 cm in diameter, and a
third 1.21 to 1.5 cm in diameter. Concomitantly, three control
cutaneous neurofibromas found on the torso and/or appendages of the
same range in sizes received sterile normal saline. All lesions
prior to injection were locally anesthetized with lidocaine 1% and
epinephrine 1:100,000. During the study period, subjects received 4
weekly intralesional injections at the 6 lesions (the 3 target
lesions receiving drug, the 3 control lesions receiving normal
saline), and were followed every week during receipt of the study
medication and three weeks after the last dose had been
administered. At every visit, subjects underwent physical
examinations, recording of adverse events and concomitant
medications, and measurement and photography of both the target and
control lesions. Assessment included the exact measured dimensions
(in two axes) of the two assessed lesions. Photography entailed
standardized digital images of both the target and control lesions
(accompanied by a ruler) without any identifying features of the
subject represented. For each subject, photography involved
consistent focal length, lighting, and angle of exposure carried
over from visit to visit. On each visit, each subject was
questioned for potential side effects of the receipt of the study
medication either during its administration or in between visits,
including pain, pruritus, irritation, discoloration, ulceration, or
any other new sign or symptom--either local or distant from the
target and control lesions--not present at baseline.
[0114] In one of the local injection studies, in which doxycycline
was the study drug, 89% of the tumors injected with doxycycline
showed either partial or complete elimination of the dermal
neurofibroma while all of the controls showed no change. Similarly,
in another study using dichlofenac as the study drug, 48% of the
tumors injected with diclofenac showed partial or complete
elimination of the dermal neurofibroma while all of the controls
showed no change.
B. Topical Administration
[0115] The subjects with neurofibromas are selected as described
above in A, and similar controls are run with untreated
neurofibromas on the same subject. Alternative methods of
administration include topical application by applying a
composition containing any one or more of the agents described
herein, in combination with 5% gel in sufficient quantities to the
skin covering a neurofibroma to cover the tumor once a day for 21
days. Additionally, topical application is performed by applying a
dermabrasion device in which the device's micro-needles are coated
with the composition comprising the agent to the skin covering the
tumor once a day for 7 days.
* * * * *
References