U.S. patent application number 12/996891 was filed with the patent office on 2011-08-11 for materials and methods for suppressing and/or treating neurofibroma and related tumors.
Invention is credited to Wade D. Clapp, David Ingram, Feng-Chun Yang.
Application Number | 20110195975 12/996891 |
Document ID | / |
Family ID | 41445287 |
Filed Date | 2011-08-11 |
United States Patent
Application |
20110195975 |
Kind Code |
A1 |
Clapp; Wade D. ; et
al. |
August 11, 2011 |
MATERIALS AND METHODS FOR SUPPRESSING AND/OR TREATING NEUROFIBROMA
AND RELATED TUMORS
Abstract
Germline mutations in the NF1 tumor suppressor gene cause Von
Recklinghausen's neurofibromatosis type 1 (NF1), a common genetic
disorder of the nervous system characterized by plexiform
neurofibroma development. Using adoptive transfer of hematopoietic
cells, we establish that NF1 heterozygosity of bone marrow derived
cells in the tumor microenvironment is sufficient to allow
neurofibroma progression in the context of Schwann cell
nullizygosity. Further, genetic or pharmacologic attenuation of the
c-kit signaling pathway in hematopoietic cells greatly diminishes
neurofibroma initiation and progression. These studies identify
haploinsufficient hematopoietic cells and the c-kit receptor as
therapeutic targets for preventing plexiform neurofibromas and
implicate mast cells as critical mediators of tumor initiation.
Administering therapeutically effective doses of a tyrosine kinase
inhibitor such as the compound imatinib mesylate to a patient in
need thereof to treat tumors in a human patient afflicted with
plexiform neurofibroma.
Inventors: |
Clapp; Wade D.;
(Indianapolis, IN) ; Ingram; David; (Indianapolis,
IN) ; Yang; Feng-Chun; (Carmel, IN) |
Family ID: |
41445287 |
Appl. No.: |
12/996891 |
Filed: |
June 25, 2009 |
PCT Filed: |
June 25, 2009 |
PCT NO: |
PCT/US09/48554 |
371 Date: |
March 24, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61076185 |
Jun 27, 2008 |
|
|
|
61103650 |
Oct 8, 2008 |
|
|
|
Current U.S.
Class: |
514/252.18 |
Current CPC
Class: |
A61P 35/00 20180101;
C07D 401/14 20130101; A61K 31/505 20130101; A61K 31/506 20130101;
A61P 25/00 20180101 |
Class at
Publication: |
514/252.18 |
International
Class: |
A61K 31/496 20060101
A61K031/496; A61P 25/00 20060101 A61P025/00; A61P 35/00 20060101
A61P035/00 |
Goverment Interests
STATEMENT OF GOVERNMENTAL RIGHTS
[0002] Part of the development of this invention was made with
government support from the NIH under grant number NS 052606 and
from the Department of Defense under grant number W81XWH-05-1-0185.
The U.S. government has certain rights in the invention.
Claims
1. A method of treating plexiform neurofibroma, comprising the
steps of: providing at least one therapeutically effective dose of
a compound according to Formula 1: ##STR00006## wherein, R.sub.1 is
4-pyrazinyl; 1-methyl-1H-pyrrolyl; amino- or amino-lower
alkyl-substituted phenyl, wherein the amino group in each case is
free, alkylated or acylated; 1H-indolyl or 1H-imidazolyl bonded at
a five-membered ring carbon atom; or unsubstituted or lower
alkyl-substituted pyridyl bonded at a ring carbon atom and
unsubstituted or substituted at the nitrogen atom by oxygen;
R.sub.2 and R.sub.3 are each independently of the other hydrogen or
lower alkyl; one or two of the radicals R.sub.4, R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 are each nitro, fluoro-substituted lower alkoxy
or a radical of formula II
--N(R.sub.9)--C(--X)--(Y).sub.n--R.sub.10 (II); wherein, R.sub.9 is
hydrogen or lower alkyl, X is oxo, thio, imino, N-lower
alkyl-imino, hydroximino or O-lower alkyl-hydroximino, Y is oxygen
or the group NH, n is 0 or 1 and R.sub.10 is an aliphatic radical
having at least 5 carbon atoms, or an aromatic,
aromatic--aliphatic, cycloaliphatic, cycloaliphatic-aliphatic,
heterocyclic or heterocyclic-aliphatic radical, and the remaining
radicals R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each
independently of the others hydrogen, lower alkyl that is
unsubstituted or substituted by free or alkylated amino,
piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or lower
alkanoyl, trifluoromethyl, free, etherified or esterified hydroxy,
free, alkylated or acylated amino or free or esterified carboxy, or
of a salt of such a compound having at least one salt-forming
group.
2. The method according to claim 1, wherein the compound is a
pharmaceutically acceptable salt of Formula 1.
3. The method according to claim 2, wherein the pharmaceutically
acceptable salt of Formula 1 is a mesylate salt.
4. The method according to claim 1, further including the step of;
diagnosing a patent with plexiform neurofibroma or a similar
condition.
5. The method according to claim 1, further including the step of;
identifying a patent at risk for developing plexiform neurofibroma
or a similar condition.
6. The method according to claim 1, wherein the therapeutically
effective dose of the compound according to Formula 1, is on the
order of between about 200 mg to about 500 mg and the dose of the
compound is administered to at patient at least once per day.
7. The method according to claim 1, wherein the therapeutically
effective dose of the compound according to Formula 1, is on the
order of between about 350 mg to about 450 mg and the dose of the
compound is administered to at patient at least once per day.
8. The method according to claim 1, wherein the therapeutically
effective dose of the compound according to Formula 1, is about 400
mg and the dose of the compound is administered to a patient twice
per day.
9. A method of treating plexiform neurofibroma, comprising the
steps of: providing at least one therapeutically effective dose of
a compound according to Formula 2: ##STR00007##
10. The method according to claim 9, wherein the compound is a
pharmaceutically acceptable salt of Formula 2.
11. The method according to claim 9, further including the step of;
diagnosing a patent with plexiform neurofibroma or a similar
condition.
12. The method according to claim 9, further including the step of;
identifying a patent at risk for developing plexiform neurofibroma
or a similar condition.
13. The method according to claim 9, wherein the therapeutically
effective dose of the compound according to Formula 2, is on the
order of between about 200 mg to about 500 mg and the dose of the
compound is administered to at patient at least once per day.
14. The method according to claim 9, wherein the therapeutically
effective dose of the compound according to Formula 2, is on the
order of between about 350 mg to about 450 mg and the dose of the
compound is administered to at patient at least once per day.
15. The method according to claim 9, wherein the therapeutically
effective dose of the compound according to Formula 2, is about 400
mg and the dose of the compound is administered to a patient twice
per day.
16. A method of treating plexiform neurofibroma, comprising the
steps of: providing at least one therapeutically effective dose of
a compound according to Formula 3: ##STR00008##
17. The method according to claim 16, further including the step
of; diagnosing a patent with plexiform neurofibroma or a similar
condition.
18. The method according to claim 16, further including the step
of; identifying a patent at risk for developing plexiform
neurofibroma or a similar condition.
19. The method according to claim 16, wherein the therapeutically
effective dose of the compound according to Formula 3, is on the
order of between about 200 mg to about 500 mg and the dose of the
compound is administered to at patient at least once per day.
20. The method according to claim 16, wherein the therapeutically
effective dose of the compound according to Formula 3, is on the
order of between about 350 mg to about 450 mg and the dose of the
compound is administered to at patient at least once per day.
21. The method according to claim 16, wherein the therapeutically
effective dose of the compound according to Formula 3, is about 400
mg and the dose of the compound is administered to a patient twice
per day.
Description
PRIORITY CLAIM
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/076,185 filed on Jun. 27, 2008 and U.S.
Provisional Patent Application No. 61/103,650 filed on Oct. 8,
2008, both of these Provisional Patent Applications are
incorporated herein by reference in their entireties as if each
were individually incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0003] Various aspects and embodiments disclosed herein relate
generally to the modeling, treatment, prevention and diagnosis of
diseases characterized by the formation of tumors, for example,
neurofibroma.
BACKGROUND
[0004] Mutations in the NF1 tumor suppressor gene cause
neurofibromatosis type 1 (NF1), a common, widely distributed human
genetic disorder that affects approximately 250,000 patients in the
US, Europe, and Japan alone. The NF1 gene encodes neurofibromin, a
320 kilodalton protein that functions, at least in part, as a
GTPase activating protein (GAP) for p21ras. Neurofibromin is highly
conserved among vertebrate species and has high homology with its
counterparts, yeast and Drosophila.
[0005] Individuals with NF1 exhibit a wide range of malignant and
nonmalignant manifestations, including plexiform neurofibromas that
collectively affect 25-40% of NF1 patients; these neurofibromas are
a major source of life long morbidity and mortality. Given the
negative impact that this condition has on people with this
mutation and the dearth of effective treatments for this and
related condition there is a pressing need for additional
treatments for this condition. Various aspects and embodiments
disclosed herein address this need.
SUMMARY OF THE INVENTION
[0006] Some embodiments include methods of treating a patient
having a form of neurofibromatosis, for example, plexiform
neurofibroma, comprising the steps of: providing at least one
therapeutically effective dose of a compound according to Formula
1:
##STR00001##
wherein, R.sub.1 is 4-pyrazinyl; 1-methyl-1H-pyrrolyl; amino- or
amino-lower alkyl-substituted phenyl, wherein the amino group in
each case is free, alkylated or acylated; 1H-indolyl or
1H-imidazolyl bonded at a five-membered ring carbon atom; or
unsubstituted or lower alkyl-substituted pyridyl bonded at a ring
carbon atom and unsubstituted or substituted at the nitrogen atom
by oxygen; R.sub.2 and R.sub.3 are each independently of the other
hydrogen or lower alkyl; one or two of the radicals R.sub.4,
R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each nitro,
fluoro-substituted lower alkoxy or a radical of formula II
--N(R.sub.9)--C(.dbd.X)--(Y).sub.n--R.sub.10 (II),
wherein, R.sub.9 is hydrogen or lower alkyl, X is oxo, thio, imino,
N-lower alkyl-imino, hydroximino or O-lower alkyl-hydroximino, Y is
oxygen or the group NH, n is 0 or 1 and R.sub.10 is an aliphatic
radical having at least 5 carbon atoms, or an aromatic,
aromatic-aliphatic, cycloaliphatic, cycloaliphatic-aliphatic,
heterocyclic or heterocyclic-aliphatic radical, and the remaining
radicals R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each
independently of the others hydrogen, lower alkyl that is
unsubstituted or substituted by free or alkylated amino,
piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or lower
alkanoyl, trifluoromethyl, free, etherified or esterified hydroxy,
free, alkylated or acylated amino or free or esterified carboxy, or
of a salt of such a compound having at least one salt-forming
group.
[0007] In some embodiments the compound is a pharmaceutically
acceptable salt of Formula 1, in some embodiment the
pharmaceutically acceptable salt of Formula 1 is a mesylate
salt.
[0008] Some other embodiments further include the step of;
diagnosing a patent with plexiform neurofibroma or a similar
condition. While still other embodiments include the step of
identifying a patent at risk for developing plexiform neurofibroma
or a similar condition.
[0009] In some embodiment the therapeutically effective dose of the
compound according to Formula 1, is on the order of between about
200 mg to about 500 mg and the dose of the compound is administered
to at patient at least once per day. In still other embodiments the
therapeutically effective dose of the compound according to Formula
1, is on the order of between about 350 mg to about 450 mg and the
dose of the compound is administered to at patient at least once
per day. In some embodiment a patient is treated twice dialing with
a therapeutically effective dose of the compound according to
Formula 1, of about 400 mg.
[0010] Some embodiments include treating a patent having a form of
neurofibromatosis, for example, plexiform neurofibroma, comprising
the steps of: providing at least one therapeutically effective dose
of a compound according to Formula 2:
##STR00002##
[0011] Still other embodiments include a method of treating a
patent having a form of neurofibromatosis, for example, plexiform
neurofibroma, comprising the steps of: providing at least one
therapeutically effective dose of a compound according to Formula
3:
##STR00003##
[0012] Still other embodiments include the use of at least one
compound according to Formulas (1), (2) or (3) or a
pharmaceutically acceptable salt thereof for the preparation of a
medicament for treating a patient having a form of
neurofibromatosis, for example. plexiform neurofibroma.
BRIEF DESCRIPTION OF THE FIGURES
[0013] The above-mentioned aspects of the present disclosure and
the manner of obtaining them will become more apparent and aspects
thereof will be better understood by reference to the following
description of the embodiments of the disclosure, taken in
conjunction with the accompanying drawings, figures, schemes,
formula and the like, wherein:
[0014] FIG. 1A. A schematic diagram of a strategy for examining the
role of the hematopoietic microenvironment.
[0015] FIG. 1B. Traces generated using fluorescence cytometry.
Nf1.sup.+/- bone marrow is necessary for plexiform neurofibroma
formation in Krox20; Nf1.sup.flox/flox and Krox20; Nf1.sup.flox/-
mice.
[0016] FIG. 2A. A Kaplan-Meier plot of percent survival (y-axis) as
a function of time (x-axis) is shown. Krox20; Nf1.sup.flox/flox and
Krox20; Nf1.sup.flox/- mice that were transplanted with Nf1.sup.+/-
or WT bone marrow were followed until 1 year of age.
[0017] FIG. 2B. Photographs of Krox20; Nf1.sup.flox/flox mice
transplanted with WT BM (1) or Nf1.sup.+/- BM (2-3).
[0018] FIG. 2C. Photographs of dissections of dorsal root ganglia
and peripheral nerves of Krox20; Nf1.sup.flox/flox mice that were
transplanted with WT or Nf1.sup.+/- bone marrow. Arrowheads
identify tumors in dorsal root ganglia and in peripheral
nerves.
[0019] FIG. 3A. Hematoxylin and eosin (H&E) sections of dorsal
root ganglia and proximal peripheral nerves.
[0020] FIG. 3B. Photographs of 200.times. magnification of sections
stained with Masson's trichrome. The genotypes of donor bone marrow
and recipient mice are indicated.
[0021] FIG. 3C. 200.times. magnification of sections stained with
Alcian blue. Small arrowheads in Panels 2 and 3 identify mast
cells. The large arrowheads in Panel 3 identify blood vessels.
[0022] FIG. 3D. Bar graph showing the difference in the number of
mast cells between different genotypes. The lineages are isolated
by FACS from tumors of Krox20; Nf1.sup.flox/flox mice transplanted
with Nf1.sup.+/- BM.
[0023] FIG. 3E. Graphic presentation of phenotypic evaluation data
of various bone marrow-derived lineages using fluorescence
cytometry. Bone marrow (panel 1) and tumor cells (panel 2) were
isolated and sorted for EGFP+ CD45.2 positive populations. Tumor
associated CD45.2 cells were further separated to identify mast
cell (panel 3), macrophage (panel 4), B-lymphocyte (panel 4) and
T-lymphocyte populations. The proportion of each hematopoietic cell
population within the tumor is indicated.
[0024] FIG. 3F. Gel showing genotyping of lineages isolated by FACS
from tumors of Krox20; Nf1.sup.flox/flox mice transplanted with
Nf1.sup.+/- BM. Arrowheads identify the amplified DNA products of
the indicated alleles from the respective phenotypic lineages.
[0025] FIG. 4A. A Kaplan-Meier plot of percent survival (y-axis) as
a function of time (x-axis) is shown.
[0026] FIG. 4B. Photographs of the spinal cord and dorsal roots of
Krox20; Nf1.sup.flox/flox mice transplanted with WT BM (panel 1) or
Nf1.sup.+/- BM (panel 2). Arrowheads identify tumors in proximal
nerves.
[0027] FIG. 4C. Graph illustrating Dorsal Root Ganglia (DRG) size
measured with donor cells of differing genotype.
[0028] FIG. 4D. Photographs of representative histologic sections
of dorsal root ganglia and proximal spinal nerves of Krox20;
Nf1.sup.flox/flox mice transplanted with Nf1.sup.+/- or
Nf1.sup.+/-; W/W mutant marrow.
[0029] FIG. 5A. PET images illustrating effects of treating of
Krox20; Nf1.sup.flox/- mice with imatinib mesylate.
[0030] FIG. 5B. Graphic summary of changes in mean FDG-PET
intensity after a 12 week treatment with imatinib mesylate or
PBS.
[0031] FIG. 5C. Graphic representation of the data collected by
dissecting certain peripheral nerves.
[0032] FIG. 5D. Photographs showing histological analysis of the
Krox20; Nf1.sup.flox/- mice treated with imatinib mesylate or
placebo.
[0033] FIG. 5E. Bar graph showing mast cell Number/HPF plotted as a
function of treatment with and without Imatnib mesylate.
[0034] FIG. 5F. Bar graph showing the number of Tunnel Positive
Cells/HPF plotted as a function of treatment with and without
Imatnib mesylate.
[0035] FIG. 6. MRI scans of a patient with plexiform neurofibromas
before and after treatment with imatinib mesylate.
[0036] FIG. 7. Ultrastructural analysis of dorsal root ganglia by
transmission electron microscopy. Panels 1-4, 750.times., Panels
5-8, 1500.times.. Panel 1-2 proximal spinal nerves from a Krox20;
Nf1.sup.flox/flox mouse transplanted with WT BM; Panels 3-8
proximal nerves from recipients transplanted with Nf1.sup.+/- BM.
(U) unmyelinated axons; (S) indicates expansion of the endoneurial
space. Arrowheads identify collagen bundles; (M) indicates mast
cells infiltrating the tumor.
[0037] FIG. 8. Photomicrographs of tissue sample taken from mice.
These images illustrate the identification of plexiform
neurofibromas using FDG-PET. FDG-PET images and dissection of
spinal nerves of a Krox20; Nf1.sup.flox/flox mouse and Krox20;
Nf1.sup.flox/- mouse imaged at 9 months of age.
[0038] FIG. 9. Photomicrographs of a tissue samples stained with
Toludine Blue and shown at 100.times. and 600.times. magnification,
the arrows point to mast cells.
[0039] FIG. 10. Gel showing genotypic identification of DNA of
individual myeloid progenitors isolated from bone marrow of
irradiated Krox20; Nf1.sup.flox/flox recipients transplanted with
Nf1.sup.+/-; Wv/Wv bone marrow.
[0040] FIG. 11A Identification of plexiform neurofibromas using
FDG-PET. FDG-PET images and dissection of spinal nerves of a
Krox20; Nf1.sup.flox/flox mouse and Krox20; Nf1.sup.flox/- mouse
imaged at 9 months of age.
[0041] FIG. 11B. Graph showing the mean intensity of FDG-PET from
the sciatic nerve region of interests in Krox20; Nf1.sup.flox/- and
Krox20; Nf1.sup.flox/flox mice.
[0042] FIG. 11C. Photographs of the representative dissections from
the dorsal root ganglia from a Krox20; Nf.sup.1flox/flox mouse
(Panel 1) and Krox20; Nf.sup.1flox/- mice with PET positive tumors
(Panels 2-4).
[0043] FIG. 12 Evaluation of apoptosis in plexiform neurofibromas
using TUNEL following treatment with imatinib mesylate or placebo.
Representative sections from plexiform neurofibromas treated with a
placebo control (left Panel) or imatinib mesylate (right Panel).
Arrowheads indicate TUNEL positive cells.
[0044] FIG. 13A. MRI images, head and neck.
[0045] FIG. 13B. Panels C, D: Axial MRI T2 weighted sequence
images; before and after 6 months of treatment with imatinib
mesylate respectively.
[0046] Corresponding reference numerals are used to indicate
corresponding parts throughout the several views.
DETAILED DESCRIPTION
[0047] The embodiments of the presented and/or described below are
not intended to be exhaustive or to limit the precise forms
disclosed in the following detailed description. Rather, the
embodiments are chosen and described so that others skilled in the
art may appreciate and understand the principles and practices of
various aspects and embodiments discussed herein.
[0048] Animals models for various diseases have proven to be very
effective tools for developing an understanding of the modeled
disease and perhaps as importantly, for developing and testing new
materials and/or methods for diagnosing, treating and/or preventing
the modeled disease. Murine models that purport to enable the
dissection of cell autonomous and non-cell autonomous contributions
to tumor development have gained considerable prominence. Rigorous
scrutiny must be exercised when extrapolating mouse tumor model to
humans and the significance of such experimental strategies can
only be appreciated in the context of a clear and verifiable
physiological relevance to the human disease state.
[0049] Accordingly, a cardinal principle in mouse modeling of human
cancer requires that mutations of relevant molecular pathways be
developed in the same tissues as the human tumors. Furthermore, the
mouse tumors used to model the human disease should recapitulate
the human phenotype in a credible series of cellular and molecular
outcomes. Provided, adequate care is taken when designing the
experiment and interpreting the results, mouse models have been
successfully used to study tumor development in humans. For
example, genetic modeling is especially fruitful when human tumors
are the consequence of a genetically inheritable trait that can be
reduced to a single gene mutation such as in the case of Von
Recklinghausens's Neurofibromatosis. Accordingly, some aspects of
the instant invention teach a mouse model suitable for minimizing
some forms of neurofibroma formation in humans. Perhaps the best
evidence that an animal model for a given disease has efficacy in
developing treatments for the same or similar diseases in humans is
to demonstrate that unlike many animal models for human diseases
treatments devised on the basis of the particular animal model are
shown to have efficacy in the treatment of human patients.
Development of a Mouse Model for Neurofibromatoris
[0050] Von Recklinghausen's Neurofibromatosis is a single gene
disorder. In the vast majority of cases it is manifested by a
germline mutation and complete somatic heterozygosity followed by a
rare loss of heterozygosity in cell types that engender the
stereotypic manifestations of this disease. Almost invariably in
this disorder, tumors develop in the peripheral nervous system of
the affected patient. Human tissue studies suggest a critical role
for Schwann cells but these studies suffer from relying on a
posteriori information to infer a preceding event. A conditional
Nf1 knockout mouse model that permits tissue specific deletion of
Nf1 has shed light on this process. In the physiologically relevant
context of global heterozygosity, these studies reveals that the
genetic bottleneck for plexiform neurofibroma formation likely lies
in the loss of Nf1 heterozygosity in the embryonic Schwann cell
lineage. The histopathological analysis of these tumors is
virtually indistinguishable from native human tumors.
[0051] The power of mouse genetics reveals a critical role for a
microenvironment that could, in distinct genetic configurations, be
either tumor permissive or tumor resistant. The data indicate that
Nf1 heterozygosity outside the Schwann cell lineage is required for
tumor formation. For example, mast cell infiltration into
peripheral nerves appears in these mice months prior to the
appearance of tumors, but not in the non tumorigenic Nf1 wild type
(.sup.flox/flox) genotype. Mast cells have been observed in human
neurofibromas, although in the absence of a mouse model,
examination of a functional role in tumor development or
maintenance could not be directly studied. It is interesting to
note that the requirement for Nf1 haploinsufficiency has been
replicated with additional Cre transgenes that target the neural
crest Schwann cell lineage including periostin-Cre, P0-Cre and
tamoxifen inducible PLP-Cre. These observations further validate
the need for Nf1 heterozygosity outside of the Schwann cell
lineage. Data gathered using tissue specific Cre transgenes do not
rule-out the possibility that tumors may develop in a wild type
environment under contrived experimental conditions. Indeed, when
we utilize neural crest specific Cre drivers that have early,
widespread, and robust expression, we observe hyperplasia of the
peripheral nerves even in .sup.flox/flox mice, although less so
than in the .sup.flox/- mice.
[0052] Results disclosed herein illustrate that the latter models
depart from the physiological situation in humans with NF1 in which
LOH is such a rare stochastic event that a nullizygous embryonic
Schwann cell precursor arising in isolation is at a relative
disadvantage in a microenvironment to develop into a tumor. These
mouse models indicate that an isolated nullizygous pocket of cells
gain a significant selective advantage by apparent synergy with
recruited heterozygous mast cells. One interpretation of these
results, consistent with the data, is that hyperplasia in robust
Cre mediated recombination models reflects a nonphysiologic
widespread loss of Nf1, not only in Schwann cells but also an
additional Nf1 loss in additional lineages that may overcome the
barriers of isolated LOH.
[0053] Neurofibromas form in association with peripheral nerves and
are composed of Schwann cells, endothelial cells, fibroblasts,
degranulating inflammatory mast cells, and pericytes/vascular
smooth muscle cells (VSMCs) and contain large collagen deposits. An
Nf1 conditional knockout mouse model confirms retrospective studies
from human tumors, demonstrating that Nf1 loss of heterozygosity
(LOH) in the Schwann cell lineage appears to be necessary but not
sufficient to elicit neurofibromas. As reported, tumor progression
requires complex interactions between Schwann cells and Nf1
haploinsufficient cell lineages in the tumor microenvironment.
Thus, in a Nf1 WT background, Nf1 nullizygosity in Schwann cells
may be necessary but not necessarily sufficient to cause tumor
formation. One reported characteristic of tumor forming
heterozygous mice is the appearance of mast cells in peripheral
nerves well in advance of tumor development. Additionally, in vitro
experiments mixing Schwann cell conditioned media and mast cells
demonstrate a hypersensitivity of Nf1 heterozygous mast cells to
conditioned media from nullizygous Schwann cells. With out wishing
to be bound by any specific theory we hypothesized a model for the
formation of those types of tumors, comprising Nf1 heterozygous
mast cell infiltration of preneoplastic peripheral nerves and
association with Nf1 nullizygous Schwann cells contributing to
tumor development.
[0054] The finding that adoptive transfer of heterozygous
Nf1.sup.+/- derived bone marrow is sufficient to cooperate with the
resident nullizygous Schwann cell lineage specific ablation to
induce tumor formation, in an otherwise WT environment, suggests
that the requirement for haploinsufficiency resides in bone marrow
derived cells but not the other cell types found in these tumors.
In addition, the genetic requirement for c-Kit signaling in the
donor bone marrow and the infiltration of donor heterozygous mast
cells into the incipient peripheral nerve tumors strongly supports
the hypothesis that this constitutes a causal interaction.
[0055] The c-Kit receptor has a central role in mast cell
development and function. Schwann cells and fibroblasts, two
principal components of neurofibromas, secrete kit-ligand in
response to many different stimuli. For example, elevated
kit-ligand mRNA transcripts have been reported in neurofibroma
tissue and it has been reported that NF 1 patients have elevated
levels of kit ligand in their serum. In preclinical trials Imatinib
mesylate, an FDA-approved pharmacological agent was thought to act
by inhibiting several tyrosine kinases including c-Kit.
[0056] When tested for efficacy against neurofibroma in a mouse
model for the disease, Imatinib mesylate exhibited an unexpected
efficacy. Administering therapeutically effective doses of the
compound has a dramatic effect on the reversal of neurofibroma
pathology. Treatment with imatinib resulted in the disappearance of
mast cells from the peripheral nerves in animals treated with this
compound. In addition to extending in vitro studies that
demonstrate c-Kit mediated interactions between NF1 Schwann cells
and heterozygous mast cells, the in vivo studies identify a
persisting requirement for imatinib mesylate responsive tumor
maintenance.
[0057] The role of inflammation and mast cells in tumor development
is an active area of research. Mast cells release mediators of
inflammation including histamine, serotonin, proteoglycans, and
leukotrienes subsequent to activation of the high affinity IgE
receptor (Fc.epsilon.RI) and the c-kit receptor. Furthermore, mast
cells reportedly release VEGF, an angiogenic factor that is also a
potent proliferative, survival, and chemotactic factor for Schwann
cells. VEGF has also been linked to an angiogenic switch in tumor
formation. Finally, mast cells also release PDGF-.beta., a growth
factor that promotes pericyte and fibroblast proliferation; and
TGF-.beta., a growth factor that promotes fibroblast proliferation
and collagen synthesis.
Imatinib Mesylate and the Tumor Microenvironment.
[0058] An important question in the context of neurofibromas is how
do mast cells cooperate with Schwann cells to elicit tumor
formation? Data presented herein indicates that other cell types in
the tumor needn't necessarily be heterozygous. However, it is too
early to assert whether the infiltrating Nf1 heterozygous mast cell
primarily acts reciprocally on the nullizygous Schwann cells to
promote tumor formation or alternatively whether indirect
interaction with local stroma and additional cell types also
present in these tumors are requisite intermediaries for tumor
induction. A better understanding of additional paracrine
interactions within neurofibromas and how they may influence tumor
formation and maintenance await additional study.
[0059] In addition, genetic results reported herein and in previous
in vitro studies demonstrated that imatinib mesylate inhibits
multiple Nf1.sup.+/- pericyte, and fibroblast tumor promoting
functions. Again, the absence of requirement for heterozygosity in
these additional tumor cells, do not rule out the possibility that
the ability of imatinib mesylate to cause the regression of tumors
may not be exclusively caused by its inhibitory activity on mast
cells. The following hypothesis is presented by way of illustration
and not limitation the fact that treatment with the compounds such
as imatinib mesylate reduces tumors is an aspect of the invention
and in no way dependent on the veracity of any given theory or
explanation proffered in an attempt to explain this result.
Imatinib mesylate and compounds of the same or similar families of
compounds may fortuitously inhibit additional and/or other
potentially critical tumor promoting activities in other tumor cell
types. For instance, in addition to inhibiting c-Kit in mast cells,
imatinib mesylate may decrease angiogenesis via PDGFR and reduce
fibrosis/collagen production via c-Abl. Additional investigation
may be required to resolve these critical and fascinating
scenarios, in the interim, one observation is that an effective
dose of this compound causes tumor regression in the mouse model
for neurofibromas and in human patients affiliated with conditions
such as plexiform neurofibromas.
Pharmacologic Inhibition of C-Kit Reduces Tumor Size and Metabolic
Activity.
[0060] Compounds that may be used to practice the present invention
include compounds of Formula 1:
##STR00004##
wherein; R.sub.1 is 4-pyrazinyl; 1-methyl-1H-pyrrolyl; amino- or
amino-lower alkyl-substituted phenyl, wherein the amino group in
each case is free, alkylated or acylated; 1H-indolyl or
1H-imidazolyl bonded at a five-membered ring carbon atom; or
unsubstituted or lower alkyl-substituted pyridyl bonded at a ring
carbon atom and unsubstituted or substituted at the nitrogen atom
by oxygen; R.sub.2 and R.sub.3 are each independently of the other
hydrogen or lower alkyl; one or two of the radicals R.sub.4,
R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each nitro,
fluoro-substituted lower alkoxy or a radical of formula II
--N(R.sub.9)--C(.dbd.X)--(Y).sub.n--R.sub.10 (II),
wherein, R.sub.9 is hydrogen or lower alkyl, X is oxo, thio, imino,
N-lower alkyl-imino, hydroximino or O-lower alkyl-hydroximino, Y is
oxygen or the group NH, n is 0 or 1 and R.sub.10 is an aliphatic
radical having at least 5 carbon atoms, or an aromatic,
aromatic-aliphatic, cycloaliphatic, cycloaliphatic-aliphatic,
heterocyclic or heterocyclic-aliphatic radical, and the remaining
radicals R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are each
independently of the others hydrogen, lower alkyl that is
unsubstituted or substituted by free or alkylated amino,
piperazinyl, piperidinyl, pyrrolidinyl or by morpholinyl, or lower
alkanoyl, trifluoromethyl, free, etherified or esterified hydroxy,
free, alkylated or acylated amino or free or esterified carboxy, or
of a salt of such a compound having at least one salt-forming
group.
[0061] The compounds of Formula 1 are generically and specifically
disclosed in the U.S. Pat. No. 5,521,184, in particular in the
compound claims and the final products of the working examples, the
subject-matter of which is herein incorporated by reference in its
entirety. In the above definition of the compound of Formula 1 the
radicals and symbols have the meanings as provided in U.S. Pat. No.
5,521,184, which is incorporated herein in its entirety.
[0062] For the purpose of the present invention, Imatinib may be
applied in the form of its mono-mesylate salt. Imatinib
mono-mesylate can be prepared in accordance with the processes
disclosed in U.S. Pat. No. 6,894,051, which is herein incorporated
by reference in its entirety. Comprised are likewise the
corresponding polymorphs, e.g. crystal modifications, which are
disclosed therein.
[0063] In adult patients a daily dose of between about 200 and
about 800 mg, e.g. 400 mg, of the mono-mesylate salt of Imatinib is
administered orally. Imatinib mono-mesylate can be administered in
dosage forms as described in U.S. Pat. No. 5,521,184, U.S. Pat. No.
6,894,051, US 2005/0267125 or WO2006/121941, all of which are
incorporated herein in their entirety as if each were separately
incorporated. For additional discussion of these compounds please
see, for example, U.S. patent application Ser. No. 11/815,046 now
U.S. publication No 2008/00114001 A1 published on May 15, 2008,
which is incorporated herein by reference in its entirety.
[0064] One especially useful compound for treatment of disease that
are thought to involve tyrosine kinase activity is the compound
imatinib mesylate
(4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-(pyridin-3-yl-
)pyrimidin-2-ylamino)phenyl]-benzamide) Imatinib can, e.g., be
prepared in accordance with the processes disclosed in WO03/066613.
One pharmaceutically acceptable salt of the compound is imatinib
mesylate shown in Formula 3 as follows:
##STR00005##
[0065] Imatinib mesylate is a potent inhibitor of the c-Kit,
PDGF-BB, and e-abl tyrosine kinases. This compound is marketing in
under the trademark protected name Gleevec. It has been approved in
the United States for the treatment of patents with Kit-expressing
(CD117+). According to the National Guideline Clearinghouse
(www.guidline.gov) the initial recommended dose level in adult
human patients in 400 mg administered twice daily. The actual
therapeutically effective dosage is patient specific and to be
determined by the prescribing physician based on various factors
including the patients weight, age, gender, age, overall health and
responsiveness to the drug.
[0066] One limitation of the mouse model is the animal's short
lifespan, given this one cannot predict on the basis of mouse
studies how long-lived tumors might be expected to respond.
However, these results with the mouse model strongly suggest that
treatment with these compounds can and will have a beneficial
effect on humans affected with some tumors. Additional support for
this hypothesis comes from the unexpectedly good result when
obtained when treatment of a human patient with imatinib mesylate
successfully reduced the size of the tumor in the human
patient.
[0067] By way of explanation and not limitation, the following
discussion is proffered. Theories concerning the cellular and
molecular mechanisms that underlie the development of idiopathic
cancer have undergone significant evolution in recent years. The
original notions that cell autonomous events convey a single cell
to overcome its normal regulation leading to development of
malignant disease, and furthermore the identity of the cell type of
tumor origin, are presently under concerted reinvestigation. It is
increasingly clear that while a series of genetic and epigenetic
events in a single cell commence the trajectory toward a malignant
phenotype, most forms of cancer include a co-option of a permissive
microenvironment that allows and even promotes the tumorigenic
state which, in accordance with this hypothesis, a resistant
environment would essentially preclude the possibility of tumor
formation. Among identified non-cell autonomous contributors to a
co-opted permissive process of tumor formation are
neo-angiogenesis, participation of the local stroma, and
inflammation among other cell types. The understanding of the
precise order of paracrine interactions, the relative importance,
and the molecular basis of the nontumorigenic environment
interaction, remains in infancy. However, the therapeutic uses of
the compounds disclosed herein are in no way limited by any of the
hypothesized modes of action or proposed molecular etiologies of
the various diseases or condition that can adventurously treated or
controlled using the materials and/or methods described herein.
[0068] These data are consistent with a role for bone marrow
derived cells in plexiform neurofibroma formation. These data also
indicates that pharmacologic and genetic inhibition of the c-kit
receptor may prevent or at least delay plexiform neurofibroma
formation in Nf1 mice. These results indicate that Nf1
haploinsufficiency of bone marrow derived cells and in particular
those dependent on activation of the c-kit receptor is required in
the tumor microenvironment to allow neurofibroma progression. The
data implicate mast cells as active participants in tumor formation
and identify novel therapeutic targets for human phase 1-2 clinical
trials.
[0069] The instant disclosure provides an example of a
physiologically relevant mouse model of a human cancer that
provides concrete insights into complex interactions between a
tumor cell of origin and the microenvironment. While,
investigations using the mouse as a model for the human disease
suggests a potential therapeutic approach for treating heretofore
untreatable tumor by targeting the microenvironment for tumor
formation rather than the tumorigenic cell. Because of the
considerable cellular and physiological differences between human
and mice, the efficacy of using compound such as imatinib mesylate
to treat neurofibroma in humans can only be "proved" by
successfully treating humans with the compound. At least in part
because humans are generally more long lived than mice, the ability
of imatinib mesylate to treat humans with conditions such as
neurofibroma, unlike the situation with the use of this compound to
treat chronic myelogenous leukemia, may lead to the development of
drug resistance. In CML imatinib mesylate is thought to act
directly on the leukemic cell and aimed at a mutated constitutively
active tyrosine kinase oncoprotein (Bcr-abl). The Bcr-abl oncogene
can develop drug resistance through the acquisition of second site
mutations. In contrast, it is likely that the principal if not
exclusive activity of anti-c-kit agents such as imatinib mesylate
on neurofibromas is on non-tumor cell acting, for example, on WT
proteins for which there may not be a ready route for the selection
for drug resistance.
[0070] We have identified compounds that in vitro have as much as
tenfold higher inhibitory activity on Nf1 heterozygous mast cells
than does imatinib mesylate (unpublished results). A very small
number of cases have been reported in which patients have both Von
Recklinghausens's Neurofibromatosis and piebaldism. These studies
inferred that piebaldism resulted from hypomorphic mutations of the
c-Kit receptor. These patients were reported to lack neurofibromas.
Another plausible explanation for the abstentions is that these
individuals were deficient in mast cells. Accordingly, these
reports may provide confirmation of the importance of c-Kit and
mast cells in neurofibroma formation in humans.
[0071] The genetic and cellular malleability of the neurofibroma
mouse models discussed here reveal important details about
microenvironment participation in tumor formation that have general
relevance for human cancer outside the context of Von
Recklinghausen's Neurofibromatosis.
Experimental
Material and Methods
Animals and Reagents.
[0072] The Krox20; Nf1.sup.flox/flox mice utilized in these studies
have been previously described (Zhu, et al., 2002), "Neurofibroma
in NF1: Schwann cell origin and role of tumor environment," Science
296, 920-922. To generate Krox20; Nf1.sup.flox/- mice, we
intercrossed Krox20; Nf1.sup.flox/flox mice with Nf1.sup.+/- mice.
All procedures involving the use of animals were approved by the
Institutional Animal Care and Use Committee at Indiana University
School of Medicine. Chemicals were purchased from Sigma (St. Louis,
Mo.) unless otherwise stated. Adoptive Transfer of Hematopoietic
Cells.
[0073] In order to evaluate the role of microenvironmental
regulation of plexiform neurofibroma progression, bone marrow
transplants were performed. Briefly, two million, syngeneic WT or
Nf1.sup.+/- bone marrow cells from WT GFP or Nf1.sup.+/- GFP mice
per recipient were adoptively transferred into young adult Krox20;
Nf1.sup.flox/flox mice and Krox20; Nf1.sup.flox/- mice after
treating them with 1100 rads of ionizing radiation administered
over two split doses.
PET Imaging Analysis.
[0074] In order to verify and anatomically locate plexiform
neurofibromas, combined [.sup.18F] fluorodeoxyglucose ([.sup.18F]
FDG) PET and x-ray CT imaging were performed. When collecting CT
images a template was placed over regions lateral to obtain on a
standardized volume of interest (VOI) thereby enabling the
researcher to quantify FDG uptake. Registered and overlaid CT image
data was used to identify specific vertebrae (e.g., landmarks from
L1 to S1). The operator then chose points along the spinal cord to
determine the path of the spinal cord through between L1 and S1.
Next, three circular regions-of-interest (ROI) are placed at
interpolated points along the spinal cord to capture the spinal
cord and the dorsal root ganglion regions. Finally, the circular
ROI's are combined to create VOI's for the spinal cord, left dorsal
root ganglion, and right dorsal root ganglion. FDG images are
acquired at 45 minutes post injection of about 0.5-1.0 mCi of FDG
via tail vein injection. All animals are given injections of FDG
while awake and isoflurane anesthesia about 40 minutes post
injection in order to immobilize the animals for imaging.
[0075] Preliminary imaging studies are conducted in non-tumorigenic
Krox 20; Nf1.sup.flox/flox mice and in Krox20; Nf1.sup.flox/- mice
that develop neurofibromas with full penetrance. In preliminary
studies both experimental PET imaging and anatomic dissection of
the dorsal root ganglia and proximal peripheral nerves failed to
detect tumors in any mice prior to 6 months of age regardless of
the genotype (data not shown). Similarly, there was little FDG
uptake in Krox20; Nf1.sup.flox/flox mice in the lumbosacral region
at 9 months of age (FIG. 8A). In contrast, an FDG PET scan of 9
month old Krox20; Nf1.sup.flox/- mice in which tumors are prevalent
in the sciatic nerve, demonstrates a specific increased FDG-PET
intensity in the lumbar region of the spine (FIG. 8A; arrows).
Subsequent necropsy of the imaged animals provided correspondent
verification of the FDG PET imaging identified tumors (FIG. 8B).
Summary FDG-PET image data from a cohort of Krox20; Nf1.sup.flox/-
mice versus a comparable age Krox20; Nf1.sup.flox/flox cohort is
shown in FIG. 8C. These studies validate noninvasive FDG-PET
imaging for identifying plexiform neurofibromas in Krox20;
Nf1.sup.flox/- mice.
Dissection of Dorsal Root Ganglia.
[0076] Immediately after sacrificing them postmortem mice are
perfused and fixed in 4% paraformaldehyde. The dorsal root ganglia
and peripheral nerves are then dissected out under a dissection
microscope. Mice whose tissue will be analyzed by electron
microscopy analysis are perfusion fixed with 2% paraformaldehyde,
2.5% glutaraldehyde, and 0.1M cacodylate (pH7.4).
Measurement of Tumor Size.
[0077] In order to evaluate a tumor's size, an anatomic measurement
of the dorsal root ganglia size is performed following measuring
the largest possible width and length of the proximal dorsal root
ganglia using a caliper. The volume of tumors is determined by
establishing the approximate volume for a spheroid (e.g.
0.52.times. (width) 2.times. length).
Phenotypic Evaluation of the Donor Cells in Dorsal Root
Ganglia.
[0078] To examine the cell type(s) from donor that reconstituted
into the tumors, flow cytometric analysis was performed. Briefly,
dorsal root ganglia was dissected out, minced, and digested by
collagenase V. The single cell suspension was then admixed with
anti CD117, CD31, or Col1A and Fc.epsilon.RI and antibodies.
Populations were then separated using a fluorescence-activated cell
sorter (Becton Dickson).
Histological Analysis.
[0079] To examine the morphology of the tumors in detail, paraffin
sections were stained with hematoxylin and eosin (H&E). Given
collagen accounts for approximately 60% of the dry weight of human
plexiform neurofibromas, the tissue sections were also stained with
Masson trichrome. To determine the existing of mast cells in the
tumors, Alcian blue staining was performed.
Transmission Electron Microscopy.
[0080] Following perfusion fixation, tissues were dehydrated in a
graded series of ethanol and acetone and embedded in Epon-Araldite
(Electron Microscopy Sciences, Hatfield, Pa.). Ultrathin sections
(silver to gold) were stained with uranyl acetate and lead citrate
and examined with a FEI Tecnai G2 electron microscope (Philips,
Eindhoven, Netherlands).
Experiment 1
Adoptive Transfer of Nf1 Heterozygous Bone Marrow (BM) Reduces
Tumor Associated Recipient Survival.
[0081] Referring now to FIG. 1, the schematic illustrates the
genotypes of recipient mice, the genotypes of adoptively
transferred cells following ionizing radiation of the recipients,
and measurements obtained following transplantation. In order to
test the hypothesis that heterozygosity of Nf1 in hematopoietic
cells within the tumor microenvironment is responsible for the
genetic haploinsufficiency required for neurofibroma formation, we
transferred Nf1 heterozygous bone marrow into mice harboring two
Krox20-Cre transgene ablated Nf1 alleles in approximately 10% of
Schwann cells (Krox20; Nf1.sup.flox/flox). Krox20;
Nf1.sup.flox/flox mice are functionally wild type in all cell
lineages and no neurofibromas are observed. As a complementary
experiment, WT bone marrow cells were transplanted into mice
containing a germline nullizygous allele of Nf1 and a foxed allele
susceptible to recombination in the Schwann cell lineage as above
(Krox20; Nf1.sup.flox/-). Krox20; Nf1.sup.flox/- mice uniformly
develop plexiform neurofibromas as previously described (Zhu et
al., 2002). A portion of the Nf1.sup.+/- or WT bone marrow was
adoptively transferred into recipients following ionizing
radiation, and the development of plexiform neurofibromas and
mortality associated with these tumors was monitored until 1 year
of age.
[0082] Referring now to FIG. 2A, the genotypes are indicated next
to each plot. Mice were sacrificed once they exhibited clear signs
of major morbidity. The Y-axis shows the percentage of the
surviving mice. Comparisons of Krox20; Nf1.sup.flox/flox+
Nf1.sup.+/- BM (dashed line) vs. Krox20; Nf1.sup.flox/flox+ WT BM
(open squares) (P<0.002); Krox20; Nf1.sup.flox/+ Nf1.sup.+/- BM
(dashed line) vs. Krox20; Nf1.sup.flox/- (open circles)--no
significant difference. Krox20; Nf1.sup.flox/- (open circles) vs.
Krox20; Nf1.sup.flox/-+WT bone marrow (closed squares)
(p<0.0.002). Six months after transplantation, the functional
germline WT (Krox20; Nf1.sup.flox/flox) recipients engrafted with
heterozygous Nf1.sup.+/- bone marrow began to exhibit motor
paralysis, weight loss and only 15% of mice survived the entire
experimental period, see, e.g., FIGS. 2A & B. This mortality
rate largely mirrored that previously observed in the germline
heterozygous Krox20; Nf1.sup.flox/- mice (FIG. 2A). In contrast,
approximately 90% of Krox20; Nf1.sup.flox/flox mice reconstituted
with WT bone marrow were alive and exhibited no clinical features
of plexiform neurofibroma formation (FIG. 2A). These data were
corroborated by the converse experiment in which transplantation of
WT bone marrow cells into germline heterozygous, Krox20;
Nf1.sup.flox/-, mice restored the survival to levels comparable to
that of non-tumorigenic Krox20; Nf1.sup.flox/flox mice that contain
intact bone marrow (FIG. 2A).
[0083] Referring now to FIG. 2C photographs of dissections of
dorsal root ganglia and peripheral nerves of Krox20;
Nf1.sup.flox/flox mice transplanted with either WT or Nf1.sup.+/-
bone marrow. Arrowheads identify tumors in dorsal root ganglia and
proximal peripheral nerves. Necropsy of the brains and spinal cords
of the morbid mice revealed that 21 of 22 Nf1.sup.+/- bone marrow
transplant recipients had an increased thickness of the entire
spinal cord as compared to the spinal cords of non-symptomatic mice
transplanted with WT bone marrow. This abnormal morphology
resembles that of the tumorigenic Krox20; Nf1.sup.flox/- mice.
[0084] Referring now to FIG. 2C, photomicrographs of the dorsal
root ganglia of mice with different genetic compositions the
arrowheads identify ganglia serving limbs that exhibit motor
paralysis. Panels 2 and 3 of FIG. 2C show the existence of discrete
tumors arising from the dorsal root ganglia of Krox20;
Nf1.sup.flox/- mice transplanted with Nf1.sup.+/- bone marrow and
these tumors were particularly prevalent in the sciatic nerves.
Volumetric analysis of the tumors revealed a 3-6 fold increase in
volume compared to unaffected dorsal root ganglia in mice that did
not develop tumors. The large size and anatomic location of the
tumors infiltrating the sciatic nerve and lumbosacral plexus,
likely account for the observed behavioral abnormalities hind limb
paralysis, hydronephrosis and enlarged, atonic bladder (FIG. 2B).
In sum, these data indicate that the presence of NF1 heterozygous
bone marrow in the context of Schwann cell loss of heterozygosity
is sufficient to recapitulate the morbidity and peripheral nerve
hyperplasia originally observed in Krox20; Nf1.sup.flox/-
tumorigenic mice.
Experiment 2
Nf1.sup.+/- Bone Marrow (BM) Recipients Develop Plexiform
Neurofibromas.
[0085] Referring now to FIG. 3A, pathologic analysis confirmed the
presence of plexiform neurofibromas in Nf1 heterozygous bone marrow
recipients Panels 1 and 6 are sections from a Krox20;
Nf1.sup.flox/flox mouse transplanted with WT BM. Panels 2, 3, 7, 8
are from Krox20; Nf1.sup.flox/flox mice transplanted with
Nf1.sup.+/- BM. Panels 4, 5, 9, 10 are from Krox20; Nf1.sup.flox/-
mice transplanted with WT BM. The photos in upper Panels were taken
with a light microscopy under 100.times., whereas the photographs
in the lower Panels were taken under 200.times.. The dorsal root
ganglia from Krox20; Nf1.sup.flox/flox mice transplanted with
Nf1.sup.+/- bone marrow exhibit classic histological features of
human plexiform neurofibromas including disruption of normal
architecture; wavy Schwann cells and infiltrating cells with
hyperchromatic nuclei (FIG. 3A, Panels 7-8); excess collagen
deposition (FIG. 3B, Panels 2,3); angiogenesis (FIG. 3C, the large
arrowheads in Panel 3 identify blood vessels); and classic
ultrastructural abnormalities (FIG. 7). In FIG. 7, Panels 1-4,
750.times., Panels 5-8, 1500.times.. Panels 1-2 proximal spinal
nerves from a Krox20; Nf1.sup.flox/flox mouse transplanted with WT
BM; Panels 3-8 proximal nerves from recipients transplanted with
Nf1.sup.+/- BM. In these photographs (U) denotes unmyelinated
axons; (S) indicates expansion of the endoneurial space; arrowheads
identify collagen bundles; and (M) indicates mast cells
infiltrating the tumor. In contrast, the nerves from either Krox20;
Nf1.sup.flox/flox or Krox20; Nf1.sup.flox/- mice transplanted with
WT marrow exhibited normal appearing, evenly distributed, nuclei
throughout sections of the dorsal root ganglia and proximal
peripheral nerves, (see, for example, FIG. 3A, Panels 1, 4-6, and
9-10) and no evidence of collagen deposition. Tumors isolated from
Krox20; Nf1.sup.flox/flox mice transplanted with Nf1.sup.+/- bone
marrow have histologic features of plexiform neurofibromas.
[0086] Referring now to FIG. 3B, Panels 1, 4-5, neovascularization,
and retained normal ultra-structural morphology (FIG. 7).
Neurofibromas are complex tumors comprising multiple cell types in
which LOH is uniquely present in Schwann cell lineage (Zhu et al.,
2002). Mast cell infiltration is characteristic of human and murine
plexiform neurofibromas (Zhu et al., 2002). In the murine model for
this condition used herein, we observe peripheral nerve
infiltration by mast cells preceding tumor appearance. Accordingly,
the heterozygous bone marrow of reconstituted Krox20;
Nf1.sup.flox/flox mice also exhibited extensive mast cell
infiltration (FIG. 3C, Panels 2-3). Fluorescence cytometry is used
to purify the endothelial cells (CD31), fibroblasts (Col1A), and
hematopoietic cells (c-Kit, CD117) in the neurofibromas of Krox20;
Nf1.sup.flox/flox mice transplanted with Nf1.sup.+/- bone
marrow.
[0087] Referring now to FIG. 3D, arrowheads identify the amplified
DNA products of the indicated alleles from the respective
phenotypic lineages. Surprisingly, subsequent genotyping showed
that only the c-Kit population, that also express Fc.epsilon.RI
(not shown), harbored the Nf1 null allele (FIG. 3D). These data are
consistent with the appearance of bona fide plexiform neurofibromas
in the reconstituted Krox20; Nf1.sup.flox/flox including homing of
the transplanted heterozygous mast cells to the sites of Nf1
nullizygous Schwann cells.
[0088] Referring now to FIG. 3E, bone marrow (BM) (band 1- and
tumor cells (panel 2) are isolated and sorted by EGFP+; CD45, 2
positive populations. Tumor associated CD45.2 cells are further
separated in order to mast cells (panel 3), macrophages (panel 4),
B-lymphocytes (panel 4) and t-lymphocytes (panel 5). The graphs
indicate the populations of each hematoporetic cell population
within the tumor.
[0089] Referring now to FIG. 3F, the genotypes of lineages isolated
by FACS from tumors of Krox20; Nf1.sup.flox/flox mice transfected
with Nf1.sup.+/- Bone Marrow (BM). The arrows in FIG. 3F, point to
bands on the gel formed by amplified DNA products of the identical
alleles isolated from the indicated phenotypic lineages.
Nf1.sup.+/- bone marrow mediated tumor formation requires
c-Kit.
[0090] The only Nf1 heterozygous cells detected in the
reconstituted plexiform neurofibromas were derived from bone
marrow. This finding is consistent with our previous in vitro and
in vivo observations implicating a mast cell haploinsufficiency
requirement in tumor formation. The c-kit receptor tyrosine kinase
(RTK) is thought to control many aspects of mast cell development
and function. We have reported that c-Kit activity appears to
govern migration, proliferation, and survival of Nf1.sup.+/- bone
marrow derived mast cells. We hypothesized a correlation between
the conditions and tested the effort of genetic disruption of c-kit
activity in Nf1.sup.+/- bone marrow cells transplanted into Krox20;
Nf1.sup.flox/flox mice on tumor progression.
[0091] Genetic disruption of c-kit in adoptively transferred
Nf1.sup.+/- bone marrow prevents the genesis of plexiform
neurofibromas in recipient Krox20; Nf1.sup.flox/flox mice. Briefly,
Nf1.sup.+/- mice were independently intercrossed with two
hypomorphic strains of mice that have inhibited mast cell
mobilization by virtue of point mutations in the c-kit receptor
that reduce kinase activity 85% (W41/W41) to 95% (Wv/Wv),
respectively.
[0092] Referring now to FIG. 4A. The bone marrow from either
Nf1.sup.+/-; Wv/Wv or Nf1.sup.+/-; W41/W41 doubly mutant mice was
transplanted into five and ten Krox20; Nf1.sup.flox/flox mice
respectively. Morbidity of mice engrafted with Nf1.sup.+/-; W
mutant marrow was significantly reduced as compared to
Nf1.sup.flox/flox mice transplanted with Nf1.sup.+/- bone marrow
Krox20; Nf1.sup.flox/flox mice were transplanted with Nf1.sup.+/-
or Nf1.sup.+/-; W mutant bone marrow were followed for 1 year. The
genotypes and statistical significance between the two groups are
indicated. Accordingly, in contrast to recipients of Nf1.sup.+/-
bone marrow, the dorsal root ganglia and proximal peripheral nerves
from the Krox20; Nf1.sup.flox/flox mice transplanted with
Nf1.sup.+/-; W mutant marrow have normal morphology and do not
exhibit hypertrophy or evidence of tumors (FIG. 4B). These results
support the hypothesis that Nf1 haploinsufficiency originates in
bone marrow and further refine the identity of the active component
to a c-Kit dependent cell type. Referring now to FIG. 4C a graph of
the volume of individual dorsal root ganglia (DRG) from the sciatic
nerves of Krox20; Nf1.sup.flox/flox mice as a function of genotype
are shown. Each individual dot represents the volume of an
individual DRG and the lines represent the mean volume from the
respective experimental groups. Recipients reconstituted with
Nf1.sup.+/- bone marrow cells have significantly higher mean
sciatic nerve DRG volumes than mice that were reconstituted with
Nf1.sup.+/- bone marrow that also contains a mutation in the c-kit
receptor that inactivates the c-kit pathway (Nf1.sup.+/-; W/W).
[0093] Referring now to FIG. 4D, the sections shown in Panels 1-3
are H&E stained. The sections in Panels 4-6 are stained with
Alcian blue. The arrow heads highlight mast cells. The genotypes of
the adoptively transferred bone marrow are indicated below the
respective columns of Panels.
Experiment 4
[0094] To assess the activity of imatinib mesylate and other
potential pharmacologic agents on tumor burden we validated a
noninvasive fluoridinated deoxyglucose positron emission tomography
FDG-PET imaging protocol permitting identification and longitudinal
observation of plexiform neurofibromas in Krox20; Nf1.sup.flox/-
mice (see also the methods section and FIG. 8).
[0095] Referring now to FIG. 5A, images change in mean FDG-PET
positive tumors after a 12 week treatment with imatinib mesylate.
The regions of interest, the temporal sequence of scans in 3
individual mice, and the experimental treatment groups are
identified. We next identified cohorts of 8 to 9 month old Krox20;
Nf1.sup.flox/- mice with confirmed PET positive uptake in the
region of the sciatic nerve for oral treatment with either 250
mg/kg/day of imatinib mesylate or a placebo control (PBS). FDG-PET
imaging studies followed to evaluate the evolution of the tumors.
Representative FDG-PET axial slices of affected nerves from three
mice imaged before and after treatment with imatinib mesylate or
PBS for three weeks are shown. As predicted, increased FDG uptake
was seen in Krox20; Nf1.sup.flox/- animals lateral to the spine
prior to treatment with either imatinib mesylate or PBS (FIG. 5A,
Panels 1, 3, 5). Strikingly, FDG uptake was qualitatively reduced
in the Krox 20; Nf1.sup.flox/- mice treated with imatinib mesylate
(FIG. 5A, Panels 2-4) compared to the PBS controls (FIG. 5A, Panel
6). To carefully quantitate the FDG-PET intensity in all treated
animals, a standardized region of interest (ROI) was utilized in
each animal to extract quantitative FDG uptake values (mCi/ml
tissue). Three dimensional ROIs in the shape of cylinders were
utilized to encapsulate the areas lateral to the spinal column with
the ROI cylinders and specific vertebrae landmarks from L1 to S1
were used in all cases to assure consistency. Representative
results from one experimental animal, before and after treatment,
are shown in Panels 7-8 (FIG. 5A). These images illustrate that
treatment with imatinib mesylate reduces tumor size in this animal
Referring now to FIG. 5B, a summary of PET imaging results
presented in graphic form for the sciatic nerve region ROIs of one
cohort of 12 experimental mice are plotted. Overall, the mice
treated with imatinib mesylate had a mean 50% reduction in FDG-PET
uptake after treatment (p<0.035). In contrast, the metabolic
activity of the tumors in the cohort treated with PBS had a modest
but not significant increase in the FDG uptake ratio comparable to
the progressive uptake in FDG observed as a function of time in
emerging plexiform neurofibromas in Krox20; Nf1.sup.flox/-
mice.
[0096] To determine whether a change in metabolic activity (FDG
uptake) directly correlated with histological changes in the
tumors, the imaged cohorts were sacrificed, the spinal cords were
examined, and sections were prepared for histologic evaluation.
[0097] Referring now to FIG. 5C, mean volume of dorsal root ganglia
from all sciatic nerves of Krox20; Nf1.sup.flox/- mice treated with
vehicle placebol vs. imatinib mesylate (n=28 in vehicle controls,
and n=28 in the gleevec treated group). The solid line indicates
the mean volume of each respective group. Each symbol indicates the
volume of an individual nerve. At post mortem, we also compared the
volumes of all dorsal root ganglia from an age equivalent cohort of
Krox20; Nf1.sup.flox/flox mice. Consistent with the FDG-PET imaging
studies, there was a clear decrease in dorsal root ganglia volume
in the imatinib mesylate treated group as compared to the PBS
treated group.
[0098] Histological evaluation of the dorsal root ganglia and
proximal surrounding nerves in mice treated with either imatinib
mesylate or PBS was also conducted. Referring now to FIG. 5D,
representative sections are shown following H&E staining,
Alcian blue staining, and Masson's trichrome staining. The
experimental therapy, the stains utilized to prepare the specimens,
is indicated on the left side of the figure. The magnification of
the images in each set of Panels is shown across the bottom of the
figure. As indicated by the column headings, samples shown in
Panels 1-6 were obtained from mice treated with imatinib mesylate;
samples shown in Panels 7-12 are obtained from mice treated with
only a placebo. The arrowheads in Panel 10 identify mast cells
found on the respective sections. Qualitatively, there is a
distinct disruption of the normal nerve architecture and an
increase in cellularity in nerves harvested from placebo treated
mice compared to imatinib mesylate treated mice. This is
illustrated in tissue sections showing the distal dorsal root and
proximal nerve segments (FIG. 5D, Panels 1-6 compared to Panels
7-12). Further, there is a marked reduction in the number of mast
cells in nerves dissected from mice treated with imatinib mesylate
compared to PBS treated controls (FIG. 5D, Panels 3, 4 vs. Panels
9, 10). Collectively, the PET, gross anatomic and histological data
identify the potential for imatinib mesylate to reduce tumor volume
in Krox20; Nf1.sup.flox/- mice.
[0099] Referring now to FIG. 5E a bar graph illustrating a dramatic
difference in the number of mast cells between mice treated with
imatinib and mice that were not treated with the compound.
Referring now to FIG. 5F, a bar graph illustrating a that there are
fewer Tunnel Positive Cells/HPF in samples taken from mice treated
with imatinib mesylate versus mice not treated with the
compound.
[0100] One limitation of our mouse model is the animal's short
lifespan. Therefore we cannot predict on the basis of our mouse
studies how long-lived tumors might be expected to respond. It is
conceivable that over time, tumor cells may accrue additional
properties that render them independent of certain early paracrine
events such as the mast Schwann cell interaction.
Experiment 5
[0101] Compassionate Use Treatment with Imatinib Mesylate of a
Child Presenting with a Plexiform Neurofibroma.
[0102] Plexiform neurofibromas primarily present in infants and
young children with NF1 are frequently characterized by rapid
growth and invasion into adjacent organs often resulting in
impairment of normal organ function. In addition, these tumors have
a high likelihood of progressing to malignancy for which there is
no cure. Even when benign, these tumors can be life threatening and
present major clinical challenges as surgical treatment has limited
effectiveness and there are no widely acceptable alternative
therapies. A three year-old child presenting the classic clinical
stigmata of NF1, including a large trigeminal plexiform
neurofibroma, presented to the pediatric oncology clinic with life
threatening airway compression. Based on our current studies in the
murine model for this and related conditions, the attending
physician placed the child on 350 mg/m2/dose of imatinib mesylate
for a limited trial.
[0103] Referring now to FIG. 6, evaluation of imatinib mesylate
efficacy in an index patient with a plexiform neurofibroma. The
periphery of the tumor is traced in both Panels by a thick dark
line. Sagital MRI scans of the head and oropharynx of an NF1
patient with a plexiform neurofibromas before (panel 1) and 3
months following treatment with imatinib mesylate (panel 2). MRI
scans before and after three months of treatment revealed a
remarkable approximately 70% reduction in tumor volume (FIG. 6).
Following treatment for six months with no observed side effects,
the patient went off treatment for six months without a recurrence
of symptoms.
[0104] Referring now to FIG. 13A. The Panels in rows A, B are
Coronal MRI T1 weighted STIR sequence images; pre-imatinib-mesylate
(row A) and 6 months following treatment with imatinib mesylate,
respectively (row B). These images demonstrate show evidence of a
profound decrease in tumor size before and after treatment.
[0105] Referring now to FIG. 13B, in both series of images, note
the marked narrowing of the upper airway (arrow) with displacement
to the right by tumor in the pre-imatinib mesylate images (row C)
versus post-imatinib treatment (the images in row D). Images
obtained after treatment with imatinib mesylate, show that there is
a marked improvement with airway enlargement back toward the
midline. The regions of the tumor in the respective images are
indicated.
[0106] This is clear evidence a pharmaceutical intervention in a
human being with the compound imatinib mesylate resulted in
significant reduction in the size of a plexiform neurofibroma.
Though reflecting the course of a single patient, this striking
result is consistent with the preclinical studies in the murine
model.
[0107] While exemplary embodiments incorporating the principles of
various aspects and embodiments have been disclosed hereinabove,
the present invention is not limited to the disclosed embodiments.
Instead, this application is intended to cover any variations,
uses, or adaptations of the information disclosed using the general
principles disclosed herein. Further, this application is intended
to cover such departures from the present disclosure as come within
known or customary practice in the art to which this invention
pertains and which fall within the limits of the appended claims
and equivalents thereof.
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