U.S. patent application number 15/229534 was filed with the patent office on 2017-01-05 for use of a neurofilament peptide for the treatment of glioma.
This patent application is currently assigned to UNIVERSITE D'ANGERS. The applicant listed for this patent is INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL UNIVERSITY, UNIVERSITE D'ANGERS. Invention is credited to Julien Balzeau, Raphael Berges, Joel Eyer, Alan Peterson.
Application Number | 20170000846 15/229534 |
Document ID | / |
Family ID | 42133636 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170000846 |
Kind Code |
A1 |
Eyer; Joel ; et al. |
January 5, 2017 |
USE OF A NEUROFILAMENT PEPTIDE FOR THE TREATMENT OF GLIOMA
Abstract
The present invention provides a new drug to treat malignant
glioma, which is the most prevalent type of primary tumor of the
central nervous system (CNS). The present invention indeed shows
that the isolated NFL-TBS.sub.40-63 peptide is highly specific for
glioma cells, in which it triggers apoptosis. It is therefore
presented here for use in a method for treating malignant glioma.
The present invention further relates to the use of the
NFL-TBS.sub.40-63 peptide for detecting specifically glioma cells
either in vivo, or in vitro, or for addressing chemical compounds
to said tumor cells.
Inventors: |
Eyer; Joel; (Blaison-Gohier,
FR) ; Peterson; Alan; (Westmount, CA) ;
Balzeau; Julien; (Segre, FR) ; Berges; Raphael;
(Angers, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITE D'ANGERS
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM)
THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL
UNIVERSITY |
Angers
Paris
Montreal |
|
FR
FR
CA |
|
|
Assignee: |
UNIVERSITE D'ANGERS
Angers
FR
INSTITU NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM)
Paris
FR
THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL
UNIVERSITY
Montreal
CA
|
Family ID: |
42133636 |
Appl. No.: |
15/229534 |
Filed: |
August 5, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13514884 |
Sep 12, 2012 |
9446092 |
|
|
PCT/EP2010/069663 |
Dec 14, 2010 |
|
|
|
15229534 |
|
|
|
|
61286207 |
Dec 14, 2009 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61K 38/16 20130101; A61P 35/00 20180101; A61K 49/0039 20130101;
A61K 49/0056 20130101 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 49/00 20060101 A61K049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 2009 |
EP |
09306227.1 |
Claims
1.-17. (canceled)
18. A method for addressing or targeting a chemical compound to
glioma cells in vivo in a subject in need thereof, or in vitro,
comprising the step of providing an amino acid sequence comprising
the NFL-TBS40-63 peptide (SEQ ID NO: 1) or a biologically active
derivative thereof.
19. The method according to claim 18, wherein said chemical
compound is a pharmaceutical compound or a labelling marker.
20. The method according to claim 18, wherein said chemical
compound is encapsulated in nanocapsules which are coupled to said
peptide.
21. The method according to claim 18, wherein said amino acid
sequence consists of the NFL-TBS40-63 peptide (SEQ ID NO: 1) or a
biologically active derivative thereof.
Description
BACKGROUND OF THE INVENTION
[0001] Malignant gliomas are the most prevalent type of primary
tumors of the central nervous system (CNS). The symptoms of a
patient with glioblastoma depend on which part of the central
nervous system is affected. A brain glioma can cause headaches,
nausea and vomiting, seizures, and cranial nerve disorders as a
result of increased intracranial pressure. A glioma of the optic
nerve can cause visual loss. Spinal cord gliomas can cause pain,
weakness, or numbness in the extremities. Gliomas do not
metastasize by the bloodstream, but they can spread via the
cerebrospinal fluid and cause "drop metastases" to the spinal
cord.
[0002] High-grade gliomas are highly-vascular tumors and have a
tendency to infiltrate. They have extensive areas of necrosis and
hypoxia. Often tumor growth causes a breakdown of the blood-brain
barrier in the vicinity of the tumor. As a rule, high-grade gliomas
almost always grow back even after surgical excision.
[0003] Gliomas can not be cured. The prognosis for patients with
high-grade gliomas is generally poor, and is especially so for
older patients. Of 10,000 Americans diagnosed each year with
malignant gliomas, only half are alive 1 year after diagnosis, and
25% after two years. Those with anaplastic astrocytoma survive
about three years. Glioblastoma multiforme (GBM) has a worse
prognosis.
[0004] Treatment for brain gliomas depends on the location, the
cell type and the grade of malignancy. Often, treatment is a
combined approach, using surgery, radiation therapy, and
chemotherapy. The radiation therapy is in the form of external beam
radiation or the stereotactic approach using radiosurgery. Spinal
cord tumors can be treated by surgery and radiation. Temozolomide
is a chemotherapeutic drug that is able to cross the blood-brain
barrier effectively and is currently being used in therapy.
[0005] Glioblastomas are the most common primary CNS malignant
glioma in adults, and account for nearly 75% of the cases. Although
there has been steady progress in their treatment due to
improvements in neuroimaging, microsurgery and radiation,
glioblastomas remain incurable. Despite the combination of surgery,
radiotherapy, and chemotherapy, the median survival of patients
with glioblastoma is limited to approximately one year, and the
five-year survival rate following aggressive therapy including
gross tumor resection is less than 10%. Glioblastomas cause death
due to rapid, aggressive, and infiltrative growth in the brain.
Failure of conventional treatments can be attributed to i) the
precarious locations of the tumors within the brain, ii) the
infiltrative nature of malignant gliomas that prevents the complete
resection of all cancer cells, and iii) the lack of specificity of
anti-tumor agents for neoplastic tissue that leads to severe
neurotoxicity.
[0006] Therefore, there is still a need for an efficient anti-tumor
drug that is able to treat gliomas, e.g. glioblastomas, without
triggering neurotoxicity.
[0007] Among antitumor drugs, antimitotic agents represent an
important class. Drugs, such as the taxane family, promote
excessive stability of microtubules. In contrast, the Vinca
alkaloids induce depolymerization of microtubules. By suppressing
microtubule dynamics or functions, such drugs lead to the
disruption of mitotic spindle function, the arrest of cell cycle
progression, and eventually apoptosis (Mollinedo et al., 2003).
[0008] WO 2005/121172 described recently that small polypeptides,
corresponding to the tubulin-binding site (TBS) and located in
intermediate filament proteins (namely the neurofilament light
chain protein NFL, keratine 8, GFAP, and vimentin) penetrate in
tumor cells (e.g. MCF7, T98G, LS187, Cos, or NGP cells) where they
disrupt the microtubule network and reduce their viability. More
particularly, Bocquet et al (2009) showed that the second
tubulin-binding site of the NFL protein (hereafter called
"NFL-TBS..sub.40-63") is able to inhibit the proliferation of
neuroblastoma and glioblastoma cell lines in vitro.
[0009] However, it was impossible, based on those results, to
anticipate the behavior and activity of NFL-TBS..sub.40-63 in vivo,
in particular on cell lines derived from malignant glioma.
[0010] Actually, it is well known that most of the chemotherapies
based on microtubule-targeting drugs fail, for the two following
main reasons: first, such drugs often result in the development of
drug resistance, mediated by overexpression of transmembrane efflux
pumps or the expression of tubulin isotypes and/or mutants that
confer resistance (Dumontet et al., 1999). Second, they lack
specificity for cancer cells and therefore induce unwanted
toxicities (Mollinedo et al., 2003). Consequently, the use of
microtubule-interacting agents has not been adapted for treating
malignant gliomas that have a less than 20% response rate to
conventional chemotherapy (Hofer et Herrmann, 2001) and for which
existing treatments are commonly associated with debilitating toxic
side effects (Cavaletti et al., 1997). A major challenge in the
field of brain tumor was thus to identify an antitumoral agent
which demonstrates therapeutic efficiency but a better specificity
than the microtubule-targeting agents for brain tumour cells over
normal tissue.
[0011] In this context, the Inventors have shown for the first time
that a microtubule-depolymerizing peptide surprisingly demonstrates
a unique specificity in vivo for glioma cells, thereby destroying
their microtubule network and inhibiting their proliferation
without obviously affecting the viability of the surrounding
healthy cells.
[0012] The results presented below reveal that, when this peptide
is injected by stereotaxy in rats bearing an intracranial F98
glioma, the size of the tumor is reduced by approximately 50%, and
the health status of animals is significantly improved.
Importantly, immunohistochemical staining revealed the presence of
the peptide only in the tumor tissue, even 24 days after its
injection, while it rapidly disappeared when injected into the same
region of the brain in normal animals.
[0013] Together, these results demonstrate a selective uptake of
the peptide used in the invention by glioma cells both in cell
cultures and in animal models, where it significantly decreases
their proliferation. Thus, it represents a promising
tubulin-binding candidate for treating malignant gliomas.
SUMMARY OF THE INVENTION
[0014] In a first aspect, the present invention relates to an
isolated amino acid sequence comprising the NFL-TBS.sub.40-63
peptide (SEQ ID NO 1), or a biologically active derivative thereof,
for use in a method for treating malignant glioma, preferably brain
malignant glioma, more preferably glioblastoma multiform (GBM).
[0015] In a second aspect, the present invention relates to the use
of an ammo acid sequence comprising the NFL-TBS.sub.40-63 peptide
(SEQ ID NO 1), or a biologically active derivative thereof, for
detecting glioma cells either in vivo, or in vitro.
[0016] In a particular embodiment, said method is a method for
testing in vitro a biological sample for the presence or absence of
malignant glioma cells, said method comprising: [0017] a.
Suspending the cells of the sample in an appropriate medium, [0018]
b. Mixing an amino acid sequence comprising the NFL-TBS.sub.40-63
peptide or a biologically active derivative thereof with the
suspended cells of the sample, [0019] c. Determining the percentage
of cells containing said amino acid sequence, wherein the
percentage of cells containing said amino acid sequence corresponds
to the percentage of glioma cells in the sample.
[0020] In a preferred embodiment, said amino acid sequence is the
NFL-TBS.sub.40-63 peptide (SEQ ID NO 1) itself.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIGS. 1A-1B demonstrate the in vitro specificity of the
penetration of the NFL-TBS40_63 peptide (10 .mu.M, 6 h) in rat
glioma cells (F98 and 9L), as compared to rat primary astrocytes
and neurons, analyzed by immunohistochemistry (FIG. 1A). Cellular
uptake of different doses of the NFL-TBS.sub.40-63 peptide (1, 5,
10, 20, 50, 100 .mu.M, 1 h, 37.degree. C.) is further analyzed by
flow cytometry (FIG. 1B).
[0022] FIGS. 2A-2B demonstrate the in vitro specificity of the
penetration of the NFL-TBS.sub.40-63 peptide (10 .mu.M, 6 h) in
human glioma cells (U87-MG and T98G) as compared to normal human
astrocytes, analyzed either by immunohistochemistry (FIG. 2A).
Cellular uptake of different doses of the NFL-TBS.sub.40-63 peptide
(1, 5, 10, 20, 50, 100 .mu.M, 1 h, 37.degree. C.) is further
analyzed by flow cytometry (FIG. 2B).
[0023] FIGS. 3A-3B show the in vitro specificity of the penetration
of the NFL-TBS4o-63 peptide (10 .mu.M, 6 h) in mouse glioma cells
(GL261) as compared to mouse astrocytes, analyzed either by
immunohistochemistry (FIG. 3A). Cellular uptake of different doses
of the NFL-TBS.sub.4o-63 peptide (1, 5, 10, 20, 50, 100 .mu.M, 1 h,
37.degree. C.) is further analyzed by flow cytometry (FIG. 3B).
[0024] FIGS. 4A-4B show the in vitro survival of rat glioma cells
(F98 and 9L) and rat primary astrocytes in the presence of various
concentration of NFL-TBS.sub.4o-63 peptide during 72 h assessed by
the MTS assay (FIG. 4A). The microtubule cytoskeleton is completely
disorganized in glioma cells but not in rat astrocytes and neurons,
as assessed by immunohistochemistry (FIG. 4B).
[0025] FIGS. 5A-5B show the in vitro survival of human glioma cells
(U87-MG and T98G) and human astrocytes in the presence of various
concentration of the NFL-TBS.sub.40-63 peptide during 72 h,
assessed by the MTS assay (FIG. 5A). The microtubule cytoskeleton
is completely disorganized in glioma cells but not in human
astrocytes, as assessed by immunohistochemistry (FIG. 5B).
[0026] FIGS. 6A-6B show the in vitro survival of the mouse glioma
cells (GL261) and the mouse primary astrocytes in the presence of
various concentration of NFL-TBS.sub.4o-63 peptide during 72 h,
assessed by the MTS assay (FIG. 6A). The microtubule cytoskeleton
is completely disorganized in glioma cells but not in mouse
astrocytes, as assessed by immunohistochemistry (FIG. 6B).
[0027] FIGS. 7A-7C show the in vitro anti-proliferative activity of
NFL-TBS.sub.40-63 peptide (100 .mu.M, 72 h) as compared with taxol
(40 nM, 72 h), NFL-SCR (100 .mu.M, 72 h) on rat cells (F98, 9L,
astrocytes, FIG. 7A), human cells (U87, T98G, astrocytes, FIG. 7B),
and mouse cells (GL261, astrocytes, FIG. 7C).
[0028] FIGS. 8A-8B reveal that the NFL-TBS.sub.40-63 peptide (100
.mu.M, 72 h) induces the in vitro apoptosis of rat glioma cells
(FIG. 8A), of human and mouse glioma cells (FIG. 8B), but not of
the corresponding astrocytes.
[0029] FIGS. 9A-9C show that the injected NFL-TBS.sub.40-63 peptide
(5 mM/60 .mu.L) selectively targets glioma cells pre-implanted in
vivo in the brain of rats, as this peptide localized on the coronal
sections only in the tumor cells, at day 16 (FIG. 9A), 24 (FIG.
9B), or 30 (FIG. 9C).
[0030] FIGS. 10A-10B show the in vivo anti-proliferative effect of
only one injection of the NFL-TBS.sub.40-63 peptide (5 mM/60 .mu.L)
on the growth of pre-implanted glioma in rat brain at days 16, 24,
and 30 on coronal sections (FIG. 10A). Quantification of the tumor
volume calculated from the coronal sections in peptide-treated or
control animals at days 16, 24 and 30 (FIG. 10B).
[0031] FIG. 11 demonstrates the therapeutical activity of only one
injection of the NFL-TBS.sub.40-63 peptide (5 mM/60 .mu.L) by
measuring the weight of the animals suffering from a glioma.
[0032] FIG. 12 shows the in vitro survival of primary human
glioblastoma cell s isolated after surgery in the presence of 100
.mu.M NFL-TBS4o-63 peptide in comparison with 100 .mu.M NFL-SCR
peptide or 40 nM taxol during 72 h assessed by the MTS assay.
[0033] FIGS. 13A-13B show that uptake of NFL-TBS.sub.40-63 peptide
is temperature and energy-dependant (FIG. 13A). (a) and (c): Glioma
cells were incubated for 30 minutes at 37.degree. C. in the 20
presence of 20 .mu.M fluorescein-tagged NFL-TBS 4o-63 peptide.
Intracellular ATP pool has been depleted (white columns) or not
(black columns) by 30 minutes of preincubation with 10 mM sodium
azide and 6 mM deoxyglucose. (b) and (d): Glioma cells were
incubated 1 hour at 37.degree. C. (black columns) or 4.degree. C.
(white columns) in the presence of 20 .mu.M fluorescein-tagged
NFL-TBS4o-63 peptide.
[0034] FIGS. 14A-D show that NFL-TBS2 peptide can be used to
improve the targeted uptake of lipid nanocapsules (LNC) in glioma
cells. GL261 cells were treated for 6 hours, and U 87-MG cells for
1 or 6 hours with different dilution of LNC containing a lipophilic
fluorochrome (DiD). Cell fluorescence was measured by FACS (FIG.
14A). Images of living GL261 cells show higher fluorescence in
cells treated with 1 O .mu.L of LNC (DiD) coupled to NFL-TBS2
peptide for 6 hours than those treated with LNC (DiD) alone (FIG.
14B). LNC (DiD)-NFL-TBS2 are both incorporated in GL261 cells (top
line) and T98G human glioma cells (bottom line) (FIG. 14C, White
bar=25 .mu.m). When LNC (DiD)-NFL-TBS2 are administered in C57B1/6
mice with GL261 tumor cells, they are sequestered in the tumor
tissue (on the right) and not in the healthy tissue (on the left)
(FIG. 14D).
DETAILED DESCRIPTION OF THE INVENTION
[0035] As mentioned previously, a major challenge in the field of
brain tumours is to identify agents that have similar therapeutic
efficiency as the microtubule-targeting agents but higher
specificity for the brain tumour cells.
[0036] The present invention discloses the surprising selectivity
of the microtubule-depolymerizing peptide NFL-TBS4o-63, which
corresponds to the second tubulin-binding site of the light
neurofilament subunit, as identified in Bocquet et al. (2009). This
peptide has been shown previously i) to inhibit microtubule
polymerization in vitro, ii) to penetrate in a human glioblastoma
cell lineage (T98G) and, iii) to disrupt the microtubule
cytoskeleton of these cells and to inhibit their proliferation
(Bocquet et al., 2009).
[0037] The NFL-TBS4o-63 peptide is 24 amino acids long and has the
following sequence: YSSYSAPVSSSLSVRRSYSSSSGS (SEQ ID NO:1). In the
context of the present application, it is referred to as the
"peptide used in the invention". As mentioned previously, it
corresponds to the second tubulin-binding site of the light
neurofilament subunit (amino acids 40 to 63 of the TBS site of the
NFL protein).
[0038] Surprisingly, the peptide used in the invention strongly
affects the proliferation of glioma cells but has poor, if not
undetectable, effects on normal astrocytes or neurons.
[0039] In contrast to traditional antimitotic agents such as taxol
or Vinca alkaloids that enter cells by passive diffusion (Gottesman
M M and Pastan I, 1993), the peptide used in the invention
penetrates selectively in glioma cells. Both immunofluorescence
microscopy and flow cytometry measures of peptide uptake revealed
in vitro a preferential uptake by glioma cells when compared to
astrocytes. Moreover, the saturable internalization demonstrated by
FACS analysis, as well as the absence of internalization of the
NFL-SCR scrambled peptide or D-amino acid peptide analogue, as well
as the absence of internalization at 4.degree. C. or in
ATP-depleted conditions, all together argue for an active and
selective transport of the NFL-TBS.sub.40-63 peptide into glioma
cells. This preferential uptake is also observable in vivo when the
NFL-TBS.sub.40-63 peptide is injected in the brain of animals
bearing or not glioma. This selective tropism of the peptide for
glioma cells when compared to other cells of the nervous system
could be due to a selective expression of cell surface-expressed
receptors by these cells. This unique property represents a major
advantage of this peptide as compared with traditional microtubule
destabilizing agents (i.e. taxanes or Vinca alkaloids), because it
results in its lack of toxicity for other cells of the nervous
system.
[0040] Therefore, in a first aspect, the present invention provides
an isolated amino acid sequence comprising the NFL-TBS.sub.40-63
peptide (SEQ ID NO:1), or a biologically active derivative thereof,
for use in a method for treating malignant gliomas.
[0041] More precisely, in this aspect, the present invention
relates to the use of an isolated amino acid sequence comprising
the second tubulin-binding site of the light neurofilament subunit
(namely NFL-TBS.sub.40-63 (SEQ ID NO 1)) or a biologically active
derivative thereof for the manufacture of a pharmaceutical
composition for treating malignant glioma.
[0042] The isolated amino acid sequence of the invention yet
comprises the NFL-TBS.sub.40-63 peptide but cannot be the entire
neurofilament light subunit itself, because this protein has not
the same biologic activity as its fragment (i.e. the
NFL-TBS.sub.40-63 peptide). In particular, the entire NFL protein
is not able to penetrate into glioma cells and has no
antiproliferative activity onto these cells. The isolated amino
acid sequence of the invention yet comprises the NFL-TBS.sub.40-63
peptide provided that it is not the entire neurofilament light
(NFL) subunit itself.
[0043] In general, the isolated amino acid sequence comprises no
more than 100 amino acids, preferably 50 amino acids.
[0044] In a preferred embodiment, the isolated amino acid sequence
of the invention consists of the NFL-TBS.sub.40-63 peptide (SEQ ID
NO 1) or a biologically active derivative thereof. Preferably, it
consists of the NFL-TBS.sub.40-63 peptide itself.
[0045] The present invention makes use of the "biologically active
derivative of the NFL-TBS.sub.40-63 peptide". As used herein, the
term "peptide derivative" includes the variants and the fragments
of the peptide to which it refers. Therefore, the "derivatives" of
the second tubulin-binding site of the light neurofilament subunit
(namely NFL-TBS.sub.40-63 (SEQ ID NO 1)) include variants and
fragments of the NFL-TBS.sub.40-63 peptide. More particularly, in
the context of the invention, the derivative designates
"biologically active" variants and fragments of this peptide, i.e.
variants and fragments retaining the biological activity and the
specificity of the parent NFL-TBS.sub.40-63 peptide. Thus, in the
context of the invention, the "biologically active" derivatives of
the NFL-TBS.sub.40-63 peptide have to show a high biological
capacity for inhibiting the proliferation of glioma cells, and have
to show a high specificity toward the glioma tumoral cells of the
brain, as the parent NFL-TBS.sub.40-63 peptide. Preferably, the
antiproliferative effect of the derivatives of the
NFL-TBS.sub.40-63 peptide on glioma cells has to be of at least
about 70%, preferably between 80% and 90%, more preferably between
90% and 99%, and even more preferably 100% of the antiproliferative
effect of the parent NFL-TBS.sub.40-63 peptide, as assessed in
vitro by conventional proliferation techniques. Also, the
derivatives of the NFL-TBS.sub.40-63 peptide have preferably the
same specificity as the parent NFL-TBS.sub.40-63 peptide toward
glioma cells, as assessed in vitro by conventional cellular uptake
experiments.
[0046] In a preferred embodiment, the derivative of the
NFL-TBS.sub.40-63 peptide is a biologically active fragment of the
NFL-TBS.sub.40-63 peptide. Said fragment comprises at least 12
successive amino acids of the parent NFL-TBS.sub.40-63 peptide,
preferably at least 16, more preferably at least 18 amino acids,
and is characterized in that it retains the biological activity and
specificity of the parent NFL-TBS.sub.40-63 peptide.
[0047] In another preferred embodiment, the derivative of the
NFL-TBS.sub.40-63 peptide is a biologically active variant of the
NFL-TBS.sub.40-63 peptide. Said variant can be either an allelic
variant of the peptide, or a peptidomimetic variant of the peptide.
An "allelic variant of the peptide" has the same amino acid
sequence as the NFL-TBS.sub.40-63 peptide, except that one or more
amino acids have been replaced by other amino acids or suppressed,
the final peptide retaining the biological activity and specificity
of the parent NFL-TBS.sub.40-63 peptide. Preferably, such allelic
variant has 70%, preferably 80%, more preferably 90% and even more
preferably 95% of identity as compared with the parent
NFL-TBS.sub.40-63 peptide (SEQ ID NO1). For example, such allelic
variant can be the TBS motif of the neurofilament light subunit of
the quail (SEQ ID NO:3), which retains 20 over 24 amino acids of
the NFL-TBS.sub.40-63 peptide. The variant of the peptide can also
be a peptidomimetic variant, which is an organic molecule that
mimics some properties of the parent peptide, including at least
one or more properties of interest that preferably is its
biological activity. Preferred peptidomimetics are obtained by
structural modification of peptides according to the invention,
preferably using unnatural amino acids, D amino acid instead of L
amino acids, conformational restraints, isosteric replacement,
cyclization, or other modifications. Other preferred modifications
include, without limitation, those in which one or more amide bond
is replaced by a non-amide bond, and/or one or more amino acid side
chain is replaced by a different chemical moiety, or one of more of
the N-terminus, the C-terminus or one or more side chain is
protected by a protecting group, and/or double bonds and/or
cyclization and/or stereospecificity is introduced into the amino
chain to increase rigidity and/or binding affinity. Still other
preferred modifications include those intended to enhance
resistance to enzymatic degradation, improvement in the
bioavailability, and more generally in the pharmacokinetic
properties, compared to the parent NFL-TBS.sub.40-63 peptide. All
of these variations are well known in the art. Thus, given the
peptide sequences of the NFL-TBS.sub.40-63 peptide, those skilled
in the art are enabled to design and produce peptidomimetics having
biological characteristics similar to or superior to such peptides.
Preferred peptidomimetic variants of the NFL-TBS.sub.40-63 peptide
retain at least the biological activity and specificity of the
NFL-TBS.sub.40-63 peptide.
[0048] The peptides used in the invention (namely the amino acid
sequence comprising the NFL-TBS.sub.40-63 peptide or its fragments,
its peptidomimetic or allelic variants) can be conveniently
synthesized using art recognized techniques.
[0049] As used herein, "percentage of identity" between two amino
acid sequences denotes the percentage of amino acids residues that
are identical between the two sequences to be compared, obtained
after the best alignment (optimum alignment), this percentage being
purely statistical and the differences between the two sequences
being distributed randomly and along their entire length. Sequence
comparisons between two amino acid sequences can be performed for
example with the BLAST program available on the website
http://www.ncbi.nlm.nih.gov/gorf/b12.html, the parameters used
being those given by default (in particular for the parameters
"open gap penalty":5 and "extension gap penalty":2, the matrix
selected being for example the "BLOSUM 62" matrix as suggested by
the program, the percentage identity between the two sequences to
be compared being calculated directly by the program).
[0050] In another embodiment, the present invention relates to a
method of therapeutically treating malignant glioma by
administering to a subject in need thereof an effective amount of a
pharmaceutical composition comprising at least an isolated amino
acid sequence comprising the NFL-TBS.sub.40-63 (SEQ ID NO 1) or a
biologically active derivative thereof.
[0051] Malignant gliomas are also known as high grade gliomas. They
can affect the brain and the spinal cord. The therapeutic method of
the invention is preferably dedicated to treat subjects carrying a
brain malignant glioma, that is chosen among anaplastic astrocytoma
(AA), glioblastoma multiform (GBM), anaplastic oligodendroglioma
(AO) and anaplastic oligoastrocytoma (AOA), and, more preferably,
carrying a glioblastoma multiform (GBM), as defined above.
[0052] In a preferred embodiment, said subject is a mammal,
preferably a mouse, a rat, a cat, or a dog, and more preferably a
human being.
[0053] Such pharmaceutical composition comprises an isolated amino
acid sequence comprising the NFL-TBS.sub.40-63 (SEQ ID NO 1) or a
biologically active derivative thereof and a pharmaceutically
acceptable carrier.
[0054] In a particular embodiment of the invention, the isolated
amino acid sequence to be incorporated in the pharmaceutical
composition of the invention is the NFL-TBS.sub.40-63 peptide (SEQ
ID NO 1) or a biologically active derivative thereof, and,
preferably, the NFL-TBS.sub.40-63 peptide itself.
[0055] In another embodiment, the peptide can be physically or
chemically linked with a radioactive moiety, a cytotoxic component,
or to an appropriate carrier (such as lipid nanocapsules as shown
in exemple 3.4. below). The peptide of the invention would address
these components specifically to the glioma cells, thereby
compromising them.
[0056] For the purpose of the invention, suitable pharmaceutically
acceptable carriers include, but are not limited to: water, salt
solutions (e.g., NaCl), alcohols, gum arabic, vegetable oils,
benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such
as lactose, amylose or starch, magnesium stearate, talc, silicic
acid, viscous paraffin, perfume oil, fatty acid esters,
hydroxymethylcellulose, and polyvinyl pyrolidone. The
pharmaceutical preparations can be sterilized and if desired, mixed
with auxiliary agents, e. g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, coloring, flavoring and/or aromatic
substances and the like which do not deleteriously react with the
active compounds. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. The composition can be a liquid solution, suspension,
emulsion, tablet, pill, capsule, sustained release formulation, or
powder. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, polyvinyl pyrollidone, sodium saccharine, cellulose,
magnesium carbonate, etc. Some appropriate precise formulations are
described, for example, in Remington, The Science and Practice of
Pharmacy, 19th edition, 1995, Mack Publishing Company.
[0057] The pharmaceutical composition can be formulated in
accordance with the routine procedures as a composition adapted for
intravenous administration to an individual. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer.
[0058] In a preferred embodiment the pharmaceutical composition of
the invention is a liquid composition that is dedicated to be
administered by intracerebral injection, and, more preferably, by
intratumoral injection. Said intratumoral injection can be obtained
for example by using stereotactic neurosurgery. This administration
can be performed prior to or after a surgical operation intended to
remove the brain tumor. In the first case, the composition enables
to inhibit the growth of the tumor and avoid dissemination of the
glioma cells and the occurrence of dramatic symptoms on the
subject; in the second case, the composition can be used to destroy
all the glioma cells that have not be removed during the surgical
operation.
[0059] The effective dose of a compound according to the invention
varies in function of numerous parameters such as, for example, the
chosen administration method, the weight, age, sex, and the
sensitivity of the individual to be treated. Consequently, the
optimal dose must be determined individually, in function of the
relevant parameters, by a medical specialist. In order to predict
the expected active doses in human from the first animal studies
presented hereunder, one can also use the k.sub.2 and C.sub.T
values as described by Rocchetti et al (2007).
[0060] It is foreseen that the effective doses for treating animals
(for example rats) range between about 0.1 micromole and 5 milimole
using a single stereotaxic injection (60 .mu.l), preferably between
about 0.2 and 0.5 micromoles. The human brain being in average 700
fold heavier than the rat brain, it is foreseen that the effective
doses for treating human glioma will range between about 0.07 and
0.7 mmol, preferably between about 0.14 mmol and 0.35 mmol. These
indicated doses are obviously to be adjusted in the context of
clinical therapeutic studies.
[0061] As shown in the experimental part of the present
application, the NFL-TBS.sub.40-63 peptide is able to penetrate
specifically into glioma cells, in vitro as well as in vivo.
Therefore, before dying from apoptosis, the glioma cells are
stained positively for the NFL-TBS.sub.40-63 peptide in a specific
manner, and can be identified among other healthy brain cells (in
particular astrocytes and neurons). The NFL-TBS.sub.40-63 peptide
is thus a promising tool to detect glioma cells, either in vitro,
or in vivo.
[0062] Therefore, in a second aspect, the present invention relates
to the use of an amino acid sequence comprising the
NFL-TBS.sub.40-63 peptide (SEQ ID NO 1) or a biologically active
derivative thereof for detecting glioma cells, preferably
glioblastoma cells.
[0063] The characteristics of the intended amino acid sequence and
of the biologically active derivative have been previously
described.
[0064] In a preferred embodiment, said amino acid sequence is
labeled so that it is easy to detect the presence or absence of the
cells containing the peptide by conventional techniques.
[0065] The term "labeled" as used herein refers to any atom or
molecule that can be used to provide a detectable (preferably
quantifiable) effect, and that can be attached to an amino acid
sequence. Labels include but are not limited to dyes, radiolabels
such as .sup.32P, binding moieties such as biotin, haptens such as
digoxygenin, luminogenic, phosphorescent or fluorogenic moieties,
mass tags; and fluorochromes alone or in combination with quenchers
that can suppress or shift emission spectra by fluorescence
resonance energy transfer (FRET). Said labels may provide signals
detectable by fluorescence, radioactivity, colorimetry, gravimetry,
X-ray diffraction or absorption, magnetism, enzymatic activity,
characteristics of mass or behavior affected by mass (e.g., MALDI
time-of-flight mass spectrometry), and the like, preferably by
fluorescence. A label may be a charged moiety (positive or negative
charge) or alternatively, may be charge neutral. Labels can include
or consist of nucleic acid or protein sequence, so long as the
sequence comprising the label is detectable.
[0066] Preferably, said labels are fluorochromes. Suitable
fluorochromes include, for example: [0067] 1. fluorescein and
derivatives, like hexachloro-fluorescein, tetrachloro-fluorescein,
carboxyfluorescein (TAMRA), CAL FLUOR.RTM. (CAL Fluor Green 520,
CAL FLUOR Gold 540, CAL FLUOR ORANGE 560, CAL FLUOR RED 590, CAL
FLUOR RED 635 available from BIOSEARCH TECHNOLOGIES), succinimidyl
ester of carboxyfluorescein (succinimidyl ester of
6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEX.TM.) or
succinimidyl ester of 6-carboxy-4',5'-dichloro-2',7'
dimethoxyfluorescein (JOE.TM.)); [0068] 2. Rhodamine and
derivatives, like 5- or 6-carboxy-X-rhodamine (ROX),
N,N,N',N'-tetramethyl-6-carboxyrhodamine; [0069] 3. Cyanine and
derivatives like Cy3, Cy3.5, Cy5, Cy5.5; [0070] 4. BODIPY.RTM.
chromophores like
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid,
4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid,
4,4-difluoro-5-styryl-4-bora-3a,4-adiaz-a-S-indacene-propionic
acid,
4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid, 4,4-difluoro-5,p-ethoxyphenyl-4-bora-3a,4a-diaza-s-indacene
3-propionic acid and
4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-S-indacene-propionic acid;
[0071] 5. Texas Red.RTM. and derivatives; [0072] 6.
Pyrenetrisulfonic acid like APTS, HPTS (CASCADE BLUE.RTM.); and
[0073] 7. Eosin and derivatives.
[0074] More preferably, said fluorochrome is selected in the group
comprising fluorescein and derivatives like hexachloro-fluorescein,
tetrachloro-fluorescein, carboxyfluorescein (TAMRA), CAL FLUOR.RTM.
(CAL Fluor Green 520, CAL FLUOR Gold 540, CAL FLUOR ORANGE 560, CAL
FLUOR RED 590, CAL FLUOR RED 635 available from BIOSEARCH
TECHNOLOGIES), succinimidyl ester of carboxyfluorescein
(succinimidyl ester of
6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEX.TM.) or
succinimidyl ester of
6-carboxy-4',5'-dichloro-2',7'dimethoxyfluorescein (JOE.TM.)).
[0075] In the context of the invention, preferred conventional
techniques to detect such fluorochrome-labeled amino acid sequence
include, but are not limited to, flow cytometry or fluorescence
microscopy.
[0076] In a preferred embodiment, the amino acid sequence of the
invention that is used to detect glioma cells consists of the
NFL-TBS.sub.40-63 peptide (SEQ ID NO 1) or a biologically active
derivative thereof.
[0077] In a more preferred embodiment, the amino acid sequence of
the invention is the NFL-TBS.sub.40-63 peptide itself, which is
coupled for example with a carboxyfluorescein dye or a biotin
tag.
[0078] In another embodiment of the invention, the present
invention relates to the use of the isolated amino acid sequence
comprising the NFL-TBS.sub.40-63 peptide or a biologically active
derivative thereof for detecting the glioma cells in vivo, or, in
other words, the isolated amino acid sequence comprising the
NFL-TBS.sub.40-63 peptide or a biologically active derivative
thereof, for its use for detecting the glioma cells in vivo.
[0079] This can be particularly useful for the medical staff to
estimate precisely the location of the tumor cells. Namely, glioma
cells are generally not located in a confined area because they are
able to infiltrate the surrounding regions of the original tumor.
Moreover, the in vivo labeling of glioma cells could help surgeons
to precisely determinate the frontier between tumor and healthy
tissues so that they can remove more completely the tumor cells
avoiding removing healthy tissues.
[0080] In this case, the amino acid sequence of the invention has
to be administered prior to a surgical operation, for example one
hour before, intracerebrally and preferably close to the tumor, so
that it penetrates inside the tumor cells and guides the surgeon in
removing all and only the tumor cells.
[0081] In this particular embodiment, the amino acid sequence of
the invention is preferably labeled with fluorescent dyes or
luminescent dyes, directly or through an appropriate carrier (such
as nanocapsules), that can be detected in safe conditions during a
surgical operation.
[0082] For example, the present invention provides a method for in
vivo detecting the presence of malignant glioma cells, said method
comprising:
[0083] a) labeling an amino acid sequence comprising the
NFL-TBS.sub.40-63 peptide or a biologically active derivative
thereof with a fluorescent or luminescent dye, directly or through
an appropriate carrier (such as nanocapsules),
[0084] b) injecting said amino acid sequence intracerebrally prior
to a surgical operation,
[0085] c) applying, during surgery, a light of particular
wave-length (depending on the fluorescent or luminescent dye) onto
the tumor region in order to reveal the glioma cells.
[0086] In another particular embodiment, the present invention is
related to the use of the isolated amino acid sequence comprising
the NFL-TBS.sub.40-63 peptide or a biologically active derivative
thereof for detecting the glioma cells in vitro, for example for
diagnosing a glioma or at least the presence of glioma cells into a
biological sample.
[0087] For example, the present invention provides an in vitro
method for testing a biological sample for the presence or absence
of malignant glioma cells, said method comprising: [0088] 1.
Suspending the cells of the sample in an appropriate medium, [0089]
2. Mixing an amino acid sequence comprising the NFL-TBS.sub.40-63
peptide or a biologically active derivative thereof with the
suspended cells of the sample, [0090] 3. Determining the percentage
of cells containing said amino acid sequence thereof, wherein the
percentage of cells containing said amino acid sequence corresponds
to the percentage of glioma cells in the sample.
[0091] As used herein, the term "biological sample" or "sample"
designates a cell culture that is handled in vitro. The cells in
culture can be either of lineage origin or of primary origin. In
this second case, the cells can be extracted from an animal brain
following a biopsy or a surgical operation.
[0092] In the in vitro method of the invention, the percentage of
cells containing said amino acid sequence "corresponds to" the
percentage of glioma cells in the sample. This means that the
percentage of cells containing the amino acid sequence used in the
invention is equivalent to the percentage of glioma cells in the
biological sample at more or less about 5%. When the absolute
number of cells in the sample is known, one can also infer from the
method of the invention the absolute number of glioma cells in the
sample at more or less about 5%.
[0093] The cells are suspended and let grown in vitro in an
appropriate medium. Such medium is well-known from a person skilled
in art and comprises advantageously glucose and L-glutamine, fetal
calf serum (e.g. 10%), and penicillin/streptomycin. The cells are
conserved in a humidified incubator gassed with 5% CO.sub.2 at
37.degree. C.
[0094] In the first step of this method, the cells are suspended,
e.g. by vortexing, so that the amino sequence of the invention can
enter in contact with all the cells of the sample.
[0095] Preferably, the concentration of the amino acid sequence to
be added is comprised between 1 and 200 .mu.M, and more preferably
between 2 and 150 .mu.M, and even more preferably between 5 and 50
.mu.M.
[0096] Preferably, the amino acid sequence is added on to the cells
during at least 30 minutes, preferably 1 hour, and then the cells
are washed extensively in order to remove the free remaining
peptide.
[0097] In this method, the amino acid sequence to be added can be
directly labeled with a dye or a conventional tag (e.g. biotin) as
previously described, or can be coupled to appropriate carrier
(such as nanocapsules). In this case, the analysis of the presence
of the amino acid sequence is performed by usual means for
detecting the dyes or tag in cellulo (e.g. flow cytometry,
immunochemistry, etc. . . . as described in the following
examples).
[0098] Alternatively, the amino acid sequence to be added is not
labeled and its detection is performed indirectly by conventional
means using for example antibodies against all or part of the amino
acid sequence. In this case, conventional techniques of indirect
detection can be used (e.g. flow cytometry, immunohistochemistry,
Western Blot, etc. . . . ).
[0099] Preferably, the amino acid sequence of the invention is
coupled to a fluorescent dye, directly or through an appropriate
carrier, and the presence in the cells is revealed by flow
cytometry (as explained in the following examples). More
preferably, the fluorescent dyes are contained in lipid
nanocapsules that are also coupled to the peptide of the
invention.
[0100] In a preferred embodiment, the in vitro method of detection
of glioma cells uses the NFL-TBS.sub.40-63 peptide itself. More
preferably, it uses a carboxyfluorescein-labelled NFL-TBS.sub.40-63
peptide or a biotin-tagged NFL-TBS.sub.40-63 peptide.
[0101] In another embodiment of the invention, the present
invention relates to the use of the isolated amino acid sequence
comprising the NFL-TBS.sub.40-63 peptide or a biologically active
derivative thereof for addressing or targeting chemical compounds
to glioma cells in vitro or in vivo. In other words, the present
invention relates to the isolated amino acid sequence comprising
the NFL-TBS.sub.40-63 peptide or a biologically active derivative
thereof, for its use for addressing or targeting chemical compounds
to the glioma cells in vivo. Also, the present invention discloses
a method for addressing or targeting chemical compounds to glioma
cells in vivo in a subject in need thereof, and a method for
addressing or targeting chemical compounds to glioma cells in
vitro.
[0102] Such chemical compounds can be directly coupled to the
peptide of the invention, or can be contained in appropriate
carriers (e.g. nanocapsules, liposomes, micelles, or any
encapsulation mean that is known by the man skilled in the art)
that are coupled to the peptide of the invention.
[0103] Said chemical compounds can be pharmaceutical compounds
and/or labeling markers such as fluorescent molecules.
[0104] Said pharmaceutical compounds are preferably cytotoxic
drugs, for example antimitotic drugs.
[0105] The chemical compounds are more preferably encapsulated in
lipid nanocapsules as described below.
[0106] In a preferred embodiment, the in vitro and in vivo methods
of targeting glioma cells use the NFL-TBS.sub.40-63 peptide
itself.
[0107] The following examples describe the high specificity and
therapeutic efficiency of the NFL-TBS.sub.40-63 peptide. They are
however not limitative, in particular concerning the nature of
amino acid sequence of the invention, and the experimental
conditions to use it.
EXAMPLES
1. Materials
[0108] Biotinylated or carboxyfluorescein-labeled peptides
corresponding to the labeled tubulin-binding site of NFL
(NFL-TBS.sub.40-63, SEQ ID NO:1) and the similarly labeled
scrambled peptide (NFL-SCR, SEQ ID NO:2) were synthesized by
Millegen (Toulouse, France), and dissolved in water at a
concentration of 1 or 5 mM. In the NFL-TBS-biotin peptide, the
biotin molecule is linked to the N-terminal end of the peptides.
Also, the carboxyfluorescein molecule is linked to the N-terminal
end of the peptides.
[0109] F98 and 9L glioma cell lines were obtained from ATCC
(Manassas, Va., USA). Cells were grown in DMEM media with glucose
and L-glutamine (Lonza France), containing 10% fetal calf serum
(Lonza France), 1% penicillin/streptomycin (Sigma) in humidified
incubator gassed with 5% CO.sub.2 (37.degree. C.) until reaching
80-90% confluence.
[0110] Rat primary astrocytes were obtained from cultures of
cerebral cortex as originally described (McCarthy and de Vellis,
1980). Briefly, the cerebral cortex was dissected from newborn rats
and cells were recovered after tissue homogenization, trypsination,
and centrifugation. They were grown during 3 weeks in DMEM media
with glucose and L-glutamine (Lonza France), containing 10% fetal
calf serum (Lonza France), 1% penicillin/streptomycin (Sigma) in
humidified incubator gassed with 5% CO.sub.2 (37.degree. C.).
[0111] Hippocampal neuronal cultures were prepared from newborn rat
brains according to Ray et al. 1993 and Kaech and Banker 2006.
Briefly, the hippocampi of animals younger than 2 days were
recovered, minced and digested in 0.01% trypsin for one hour at
37.degree. C. Dissociated cells were plated on coverslips precoated
with 5 .mu.g/ml poly-l-lysine and 7 .mu.g/ml collagen at densities
of 2.times.10.sup.5/ml and incubated at 37.degree. C. with a 5% CO2
atmosphere. Twenty-four hours later, the plating solution was
replaced by B-27 neurobasal medium, and the second day cytosine
arabinoside (20 .mu.M) was added to eliminate non-neural cells.
Experiments were performed 7 days after plating.
[0112] Human glioblastoma cell lines of U87-MG and T98G were
obtained from ATCC (Manassas, Va., USA). GL261 mouse glioma cell
line was kindly provided by Dr P Walker (Laboratory of Tumor
Immunology, University Hospital Geneva, Switzerland). Human
astrocytes were obtained from Lonza France. Purified newborn mouse
primary astrocytes were obtained by the mechanical dissociation
method from cultures of cerebral cortex as originally described
(McCarthy and de Vellis, 1980).
[0113] Human glioblastoma cells, GL261 glioma cells and mouse
primary astrocytes were grown in DMEM media with glucose and
L-glutamine (Lonza France), containing 10% fetal calf serum (Lonza
France), 1% penicillin/streptomycin (Sigma) in humidified incubator
gassed with 5% CO.sub.2 (37.degree. C.) until reaching 80-90%
confluence. Human astrocytes were cultured in Astrocyte Basal
Medium (ABM) (Lonza) supplemented with the AGM SingleQuots growth
factor (Lonza). Cells were maintained according to manufacturer's
instructions.
[0114] Primary human glioblastoma cells obtained from tissue
samples extracted in human brains during surgery are put in culture
in a DMEM medium containing glucose, L-glutamine, 10% fetal calf
serum, 10% penicillin/streptomycin (Sigma). Cells were plated (20
000 cells per cm.sup.2) in MW96 and let grow at 37.degree. C. in a
humidified incubator gassed with 5% CO.sub.2. The peptides and/or
drugs are added 48 hours after their plating.
[0115] Lipid nanocapsules (LNC) were performed as previously
described (Heurtault et al., 2002). Briefly, Solutol HS-15, Lipoid
S75-3, sodium chloride, Labrafac CC and water were mixed by
magnetic stiffing (200 rpm) at room temperature leading to an
oil/water emulsion. After heating, an interval of transparency at
70.degree. C. can be observed, and the inverted phase "water in
oil" is obtained at 85-87.degree. C. Three temperature cycles
alternating from 60 to 87.degree. C. were applied, then before the
last decrease of temperature, the mixture was diluted with 12.5 mL
of cold water (close to 0.degree. C.) and stirred for 15 min. DiD
was added just before the last dilution. After LNC
characterization, 369 .mu.L of NFL-TBS2 (1 mM) were added to 1 mL
of LNC (DiD) for peptide adsorption during 24 hours at 18.degree.
C. Then, a new characterization of LNC was performed.
2. Methods
2.1. Analysis of Cell Viability and Proliferation
[0116] The effects of peptides on the proliferation of glioma cells
or astrocytes were evaluated by the MTS cytotoxicity assay and by
counting directly the number of cells. For the MTS assay (Promega),
500 cells were seeded in 96-well plates, incubated at 37.degree. C.
for 24 hours, and treated by the peptide at the indicated
concentrations for 24, 48 and 72 hours, or with vehicle (PBS or
water). Media and peptides were replaced daily. Peptides were
prepared in DMEM, and paclitaxel was dissolved in DMSO at a
concentration of 2 mM and further diluted into DMEM. Viability was
also determined by trypan blue staining. For manual counts, cells
were treated with 0.25% trypsin/0.53 mM EDTA, centrifuged, and
counted with a hemacytometer following addition of trypan blue dye.
At each time, 3 to 6 wells per treatment were counted.
[0117] To assess cell proliferation, 5-bromodeoxyuridine (BrdU)
immunohistochemistry was used. Cells were platted on coverslips and
cultured in media containing biotinylated peptides (100 .mu.M) for
72 hours, and incubated during 4 hours in the presence of 1 mg/mL
BrdU (Sigma). Cells were then washed in PBS, fixed in 3%
paraformaldehyde for 10 min and permeabilized with 1% Triton X-100
in PBS for 10 min. Cells were acidified to denature the DNA (2 N
HCl, 10 min), neutralized (0.1 M sodium borate, 10 min) and then
rinsed extensively in PBS. After blocking with 10% NGS (10 min) the
cells were labeled with monoclonal anti-BrdU antibody ( 1/400)
followed by Alexa 568 nm anti-mouse antibody ( 1/200). Nuclei were
stained with 4'6-diaminido-2-phenylindole (DAPI; Sigma). The
stained cells were observed with a Leica DMI6000 inverted
microscope. Images were acquired with a CoolSNAP HQ2 camera and
analyzed with Metamorph 7.1.7.0. software. Minimums of 200 cells
were scored for BrdU incorporation, and experiments were repeated
at least three times.
2.2. Cellular Uptake Analysis by Flow Cytometry
[0118] To evaluate the internalization of fluorescein-labeled
NFL-TBS.sub.40-63 peptide by FACS, glioma cells or astrocytes were
seeded in 35 mm dishes and cultured for 1 hour at 37.degree. C. in
media containing fluorescein-labeled NFL-TBS.sub.40-63 peptide at
increasing concentrations or with vehicle (PBS). Cells were
trypsinized, washed twice in cold PBS before incubation with
trypsin (1 mg/mL) during 15 min at 37.degree. C. To investigate a
possible energy-dependant mechanism for the uptake of the peptide,
cells were incubated at 4.degree. C. with 20 .mu.M
fluorescein-labeled NFL-TBS2 peptide (after 15 min of 4.degree. C.
preincubation), or with 10 mM sodium azide in the presence of 6 mM
2-deoxy-D-glucose for 1 hour to deplete cellular ATP before
addition of 20 .mu.M fluorescence-labeled NFL-TBS2 peptide. Cells
were then washed once, resuspended in 500 .mu.l containing 50
.mu.g/mL propidium iodide (Sigma, Saint-Quentin Fallavier, France),
and analyzed with FACScalibur flow cytometer. Experiment on each
cell type was repeated three times. 20,000 events per sample were
analyzed in each experiment.
2.3. Cell Death Analysis by Flow Cytometry
[0119] To detect possible apoptotic processes, cells were seeded in
35 mm dishes and cultured in media containing biotinylated peptides
(100 .mu.M) or PBS alone for 72 hours. Paclitaxel (40 nM) was used
as a positive control to induce apoptosis (Terzis et al., 1997).
Cells were then trypsinized, washed in cold PBS, and stained with
Annexin V-FITC (BD Pharmingen) in Annexin buffer for 15 min at room
temperature. Finally, they were counterstained with 50 .mu.g/mL
propidium iodide (Sigma) and analyzed with a FACSCalibur flow
cytometer. Experiment on each cell type was repeated three times.
20,000 events per sample were analyzed in each experiment.
2.4. Immunocytochemistry
[0120] Cells were plated on coverslips and cultured in media
containing biotinylated peptides (10 .mu.M) for 6 hours. Following
PBS washing, the cells were fixed for 10 min in 4%
paraformaldehyde, and washed 3 times in PBS. They were then
incubated for 10 min in a 0.5% triton X-100 permeabilization
solution, washed 3 times in PBS before incubation in a blocking
solution (5% BSA) for 15 min. Glioma cells and astrocytes were
incubated overnight at 4.degree. C. with mouse anti-B-tubulin
antibody (Sigma) 1/200, and neurons with mouse
anti-.beta.III-tubulin antibody 1/200. Tubulin and biotinylated
peptides were localized using respectively Alexa 568 nm anti-mouse
antibody and streptavidin Alexa 488 nm (Molecular Probes) 1/200 for
1 hour, followed by washing in PBS. The preparations were
counterstained with 3 .mu.M 4'6-diaminido-2-phenylindole (DAPI;
Sigma) for 5 min, and washed twice with PBS. Coverslips were
mounted with an antifading solution.
[0121] Observations were carried out with an Olympus confocal
microscope (BX50) using Fluoview.3.1. Software or a Leica DMI6000
inverted microscope. Images were acquired with a CoolSNAP-HQ2
camera and analyzed with Metamorph 7.1.7.0. software. Cells that
are positive for peptide staining and cells with destroyed
microtubule network were counted. Experiments on each cell type
were repeated at least three times, and minimums of 200 cells were
analyzed for each experiment.
2.5. Animal Studies
[0122] 9 to 10 weeks old female syngeneic Fisher 344 rats were
obtained from Charles River Laboratories France (L'Arbresle,
France). The animals were housed in a temperature and
humidity-controlled room with 12-hour on-off light cycles, and
given free access to food and water.
[0123] All experimental procedures and animal care were carried out
in conformity with the guidelines of the French Government and
approved by the Regional Committee for Ethics on Animal
Experiments.
[0124] Rat F98 cells at 70% confluency were trypsinized, counted on
an hemacytometer, and checked for viability by trypan blue
exclusion. Cells were washed twice in DMEM and a final suspension
of 5.times.10.sup.4 cells/mL in DMEM was obtained. Animals were
anesthetized by intraperitoneal injection of a mixture of ketamine
10% (0.8 .mu.l/g), and xylazine 2% (0.5 .mu.l/g). Using a
stereotaxic frame (David Kopf instruments, Tujunga, Calif., USA), a
sagital incision was made through the skin to expose the cranium,
and a small dental drill was used to make a burr hole in the skull
1 mm anterior and 2 mm lateral to the bregma. A volume of 10 .mu.l
of DMEM alone or containing 500 tumor cells was injected at a flow
rate of 2 .mu.l/min using a 10-.mu.l Hamilton syringe (Hamilton
glass syringe 700 series RN) with a 32-G needle (Hamilton), at a
depth of 4 mm deep from the outer border of the cranium into the
striatum of the rat. The needle was left in place for an additional
5 min to avoid expulsion of the suspension from the brain, and then
slowly withdrawn (0.5 mm/min).
[0125] Six days following glioma implantation, 60 .mu.l infusions
were performed at the coordinates of the tumor cells using a
10-.mu.l Hamilton syringe with a 32-G needle. This syringe was
connected to a 100-.mu.l Hamilton 22-G syringe containing the
peptide (Harvard Apparatus, Les Ulis, France) through a cannula
(CoEx.TM. PE/PVC tubing, Harvard Apparatus). Slow-infusion
Convection-Enhanced Delivery procedure (CED) was performed with an
osmotic pump (Harvard Apparatus) at a rate of 0.5 .mu.l/min for 2
hours to achieve a total volume of 60 .mu.l (Reference). After
infusion, the needle was removed and the wound sutured.
[0126] Following intracerebral tumor cell implantation (day 0),
rats were randomized into 4 groups. Six days post-implantation (day
6), the rats were treated by CED as follows: group 1: controls (60
.mu.l of vehicle; n=10); group 2: 60 .mu.l of 1 mM
NFL-TBS.sub.40-63 peptide (n=7); group 3: 60 .mu.l of 1 mM NFL-SCR
peptide (n=7); group 4: 60 .mu.l of 5 mM NFL-TBS.sub.40-63 peptide
(n=7).
[0127] The animals were monitored each day for their clinical
status (weight loss, ataxia, and periorbital hemorrhage) (Redgate
et al., 1991). They were euthanized when affected by hemiplegia or
at least 20% of weight loss. Animals were sacrificed at day 16, day
23, and day 30 (n=3/group) and their brain was removed, frozen in
isopentane at -30.degree. C., and stored at -80.degree. C.
[0128] Frozen brains were serially sectioned using a Leica
cryostat, and 20 .mu.m sections were HE-stained for histomorphology
and measure of the tumor volume. Images of HE-stained sections were
captured with a Leica Z16APO macroscope using the Leica Application
Suite 2.8.1 Software. The tumor area was manually outlined and
measured using Image J software. Knowing the thickness and the
number of sections, the total volume of each tumor was calculated.
Tumor volumes were measured for three animals per group.
[0129] For immunohistochemistry, 12 .mu.m sections were fixed with
cold methanol during 10 min, washed 3 times in PBS, before blocking
at room temperature for one hour with PBS 5% BSA. Sections were
incubated with mouse anti-GFAP antibody (Sigma) 1/200 in PBS 5% BSA
overnight, and then rinsed with PBS (3.times.5 minutes). GFAP and
biotinylated peptides were localized using respectively anti-mouse
antibody Alexa 568 nm and streptavidin Alexa 488 nm (Molecular
Probes), diluted 1/200 in PBS 5% BSA for 90 minutes, followed by
washing in PBS. The preparations were counterstained with 3 .mu.M
4'6-diaminido-2-phenylindole (DAPI; Sigma) for 5 min and washed
twice with PBS. Slides were mounted with an antifading solution and
observed with a Leica DMR fluorescence microscope and the Leica
IM500 software.
[0130] MRI and .sup.1H-magnetic resonance spectroscopy: MRI was
performed with a Bruker Avance DRX 300 (Germany) apparatus equipped
with a vertical superwide-bore magnet of 7T. Qualitative
T2-weighted images were obtained using rapid acquisition with
relaxation enhancement (RARE) sequence (TR=2,000 ms; mean echo time
(Tem)=31.7 ms; RARE factor=8; FOV=3.times.3 cm; matrix
128.times.128; nine contiguous slices of 1 mm, eight acquisitions).
Magnetic resonance spectroscopy (MRS) was performed using a PRESS
sequence with water suppression and cardiac triggering (Rapid
Biomed GmbH, Germany). .sup.1H spectra were acquired with the
following parameters: TR/TE=1,500/11 ms; NEX=128; vowel size 27
.mu.l (3.times.3.times.3 mm).
2.6. Statistical Analysis
[0131] Data are presented as mean S.E.M. (bars). Cell counting,
cellular viability data, tumor volumes were analyzed by Student's t
test using Prism version 3.00 (GraphPad Software, San Diego,
Calif.). Asterisks indicate significant level vs. control*,
p<0.05; **, p<0.005; ***, p<0.0001.
3. Results
3.1. Specificity of NFL-TBS.sub.40-63 for Glioma Cells in Vitro
3.1.1. Specificity of the Penetration
[0132] To evaluate the specificity of NFL-TBS.sub.40-63 (10 .mu.M)
on different cell types from the brain, its effects on rat F98 and
9L glioma cells, as well as primary cultures of rat astrocytes and
neurons, was analyzed by immunofluorescence microscopy.
[0133] Image analysis and cell counting showed that more than 50%
of the total cells contained detectable NFL-TBS.sub.40-63 peptide
(53.5%.+-.1.5 for F98, and 58.2%.+-.9 for 9L). At similar doses,
the peptide penetrates much less in astrocytes (9%.+-.4.6) or
neurons (17.9%.+-.5.9) (FIG. 1A).
[0134] Interestingly, similar results were obtained with human and
mouse derived cells (FIG. 2A and FIG. 3A respectively).
[0135] Fluorescent-activated cell sorter (FACS) measurements were
performed to further quantify cellular uptake of
carboxyfluorescein-tagged peptides. To discriminate between
membrane-bound and internalized fluorochrome, trypsin treatment of
the cells before FACS analysis has been performed to avoid
surface-binding of the peptide. Following incubation of the cells
during 1 hour with 50 .mu.M of peptide, 89.6%.+-.2.5 of F98 and
100%.+-.0 of 9L glioma cells contained the NFL-TBS.sub.40-63
peptide, while only 28.0%.+-.2.5 of astrocytes are positive (FIG.
1B).
[0136] These data show a preferential penetration of the
NFL-TBS.sub.40-63 peptide in rat glioma cells.
[0137] Interestingly; similar results were also obtained with human
and mouse malignant glioma cells (FIG. 2B and FIG. 3B
respectively).
[0138] When glioma cells were incubated with the peptide at
4.degree. C., or when ATP pool was depleted by preincubation of the
cells with sodium azide and deoxyglucose, a significantly reduced
uptake of NFL-TBS2 was observed indicating an energy dependant
mechanism of internalization (FIG. 13A). Moreover, when T98G cells
are treated with 10 .mu.M of D-amino acid peptide analogue and
analyzed by immunocytochemistry, internalization of peptide is
strongly reduced, suggesting a receptor-mediated internalization by
endocytosis (FIG. 13B).
[0139] Finally, the peptide also penetrates specifically in primary
human glioblastoma cells isolated after surgery (data not
shown).
[0140] All together, these in vitro results show importantly that
the NFL-TBS.sub.40-63 peptide penetrates specifically in glioma
cells of human, rat, and mouse origin, in cell lineages as well as
in primary glioma cells. On the contrary, these results point out
that the NFL-TBS.sub.40-63 peptide do not enter into the non
tumoral cells present in the brain, that is astrocytes and neurons.
This result will be further confirmed in the in vivo model of rats
bearing F98 glioblastoma (see point 3.2. of the examples).
3.1.2. Reduced Viability of Malignant Glioma Cell in the Presence
of NFL-TBS.sub.40-63
[0141] Rat glioma cells (F98 and 9L) and astrocytes were treated
with the NFL-TBS.sub.40-63 peptide or its control scrambled
sequence (NFL-SCR) at different concentrations (0, 20, 50 and 100
.mu.M) and during different times (24, 48 and 72 hours). Taxol (40
nM) was used as a positive control for cytotoxicity. The MTS
cytotoxicity assay, based on the capacity of viable cells to
convert MTS to formazan by their mitochondrial dehydrogenase
enzymes, was used. It was found that the cell viability of the two
rat glioma cell lines was significantly reduced by 60.8%.+-.2.8 for
F98, and 30.0%.+-.4.4 for 9L following 72 hours treatment with 100
.mu.M of the NFL-TBS..sub.40-63 peptide (FIG. 4A).
[0142] These results were reproduced using human and mouse
malignant glioma cells (see FIGS. 5A and 6A respectively).
[0143] Moreover, the cell survival of primary human glioblastoma
cells isolated after surgery is also greatly affected by the
NFL-TBS.40-63 peptide: after 72 hours of incubation with the
NFL-TBS..sub.40-63 peptide (100 .mu.M), the cell survival is
reduced by 50%. Interestingly, the cell survival is at the same
level when the cells are treated with 40 nM of taxol, suggesting
that the NFL-TBS..sub.40-63 peptide has at least the same effect on
cell viability than this well-known microtubule-depolymerizing drug
(see FIG. 12).
[0144] Cell viability was also evaluated by Trypan blue dye
exclusion test. This negatively charged chromophore only penetrates
in cells when their membrane is damaged. In consequence, all cells
that exclude the dye are viable. It has been observed that the
NFL-TBS.sub.40-63 peptide strongly affected the viability of rat,
human and mouse gliomas, while astrocytes were not affected (data
not shown).
[0145] To study the mechanism that potentially explains the reduced
cell viability of the glioma cells in presence of the
NFL-TBS..sub.40-63 peptide, the microtubule cytoskeleton of these
cells has been assessed by immunohistochemistry.
[0146] While untreated F98 and 9L cells were large and filled with
a dense network of microtubules, those containing the
NFL-TBS..sub.40-63 demonstrated an atypical spherical shape with
their tubulin co-aggregated with the intracytoplasmic peptide (not
shown). Such alterations were observed in 82%.+-.3 of total F98
cells and 76.7%.+-.5.8 of total 9L cells. In contrast, the
NFL-TBS..sub.40-63 peptide only poorly affected astrocytes
(4.7%.+-.0.6), and neurons (8.5%.+-.1.5). Moreover, the scrambled
peptide NFL-SCR didn't alter the microtubule network of glioma
cells, astrocytes and neurons (FIG. 4B). Similar results were
obtained with human and mouse derived cells (FIGS. 5B and 6B
respectively).
[0147] All together, these results show that the NFL-TBS.sub.40-63
peptide destroys the microtubule cytoskeleton and reduces the
viability of glioma cells, either from human, rat or mouse origin,
whereas it does not affect the microtubule cytoskeleton and the
viability of non tumoral cells that are also present in the brain,
for example astrocytes and neurons. Importantly, this result has
also been reproduced in vivo in non tumor-bearing (control) rats,
in which the peptide had basically no effect and is rapidly
eliminated (see 3.2. of the examples).
3.1.3. Specificity of the Anti Proliferative Effect
[0148] MTS assays and Trypan blue tests have shown that the
NFL-TBS.sub.40-63 peptide induced a decrease of the number of
living cells. In order to determine whether this reduction reflects
1) a decrease of the cell proliferation (cytostatic effect), 2) a
toxicity leading to cell death (cytotoxic effect), or 3) a
combination of these two effects, the impact of peptide on the
proliferation of the different cell types (rat glioma cell lines
and astrocytes) was analyzed by measuring the incorporation of
bromodeoxyuridine (BrdU), a thymidine analog, into DNA. This
reveals the number of cells in S phase during the BrdU treatment
and thereby the number of proliferating cells.
[0149] The treatment of rat glioma cells with 100 .mu.M of
NFL-TBS.sub.40-63 peptide strongly decreased the incorporation of
BrdU in these two lines examined (78.2%.+-.3.0 inhibition of F98
cells, and 34.8%.+-.2.6 of 9L cells) (FIG. 7A), indicating a lower
number of proliferative cells. In strong contrast, a similar
treatment of rat astrocytes did not affect their proliferation.
Moreover, the NFL-SCR peptide has no effect on all these cells.
[0150] Similar results were obtained when cells derived from human
and mouse were tested (FIGS. 7B and 7C respectively).
[0151] To conclude, these results show that the NFL-TBS.sub.40-63
peptide reduces specifically the viability of glioma cells, either
from human, rat or mouse origin. On the contrary, it does not
affect the viability of non tumoral cells that are also present in
the brain, for example astrocytes.
3.1.4. Specific Induction of Apoptosis in Malignant Glioma
Cells
[0152] BrdU staining analysis demonstrated a cytostatic effect of
the NFL-TBS..sub.40-63 peptide on glioma cells. Microtubule-binding
drugs are known to arrest proliferation and to induce cell death by
apoptosis. To examine whether the decreased number of living glioma
cells observed with the MTS assay and Trypan blue test was also
associated with cell death (cytotoxic effect), F98, 9L glioma cells
or astrocytes cells were incubated with 100 .mu.M of the peptide
during 72 hours, and then harvested and stained with propidium
iodide (PI) and annexin V. Then, apoptosis quantification was
evaluated by FACS analysis. Viable cells with intact membranes
exclude PI, whereas the membranes of dead and damaged cells are
permeable to PI. Moreover, the membrane phospholipid
phopsphatidylserine (PS) is translocated from the inner to the
outer leaflet of the external cellular environment in apoptotic
cells, and thus Annexin V protein staining (which has a high
affinity for PS and binds to cells with exposed PS) can be used to
detect apoptotic cells.
[0153] As shown in FIG. 8A, following NFL-TBS.sub.40-63 treatment,
the percentage of F98 cells in early or late apoptotic stage (which
are considered as dead cells), was respectively 233.5%.+-.43.3 and
347.7%.+-.32.6 higher than negative control. Similarly, 9L cells
displayed an increased number of early apoptotic (712.1%.+-.581.3)
and late apoptotic (233.6%.+-.82.3) following such a treatment.
Apoptotic response to NFL-TBS.sub.40-63 peptide was correlated with
the significant decrease of the living cell number in both F98
(-37.6%.+-.5.0) and 9L (-32.6%.+-.7.4) gliomas, in comparison with
negative control. In contrast, primary astrocytes were not
sensitive to NFL-TBS.sub.40-63 peptide. At the same concentration,
the NFL-SCR peptide has no effect on these different cell
types.
[0154] Similar results were obtained when cells derived from human
or mouse were tested (FIG. 8B).
[0155] To conclude, these results demonstrate that the inhibiting
effect of the NFL-TBS.sub.40-63 peptide on glioma cell
proliferation is mediated by an active apoptosis mechanism, a cell
death mechanism shared by various cancer cells, especially
glioblastoma cells, when treated with antimitotic drugs (Wang et
al., 2000). In other words, the NFL-TBS.sub.40-63 peptide is
therefore able to induce the death of glioma cells by apoptosis but
has no effect on normal healthy brain cells.
3.2. Specificity of the Penetration of NFL-TBS.sub.40-63 in Glioma
Cells in Vivo
[0156] Experiments in vitro showed a selective uptake of the
NFL-TBS.sub.40-63 peptide by glioma cells when compared to
astrocytes or neurons. It was further tested whether the peptide
could also target only tumor cells in vivo and inhibit selectively
their proliferation using rats bearing F98 gliomas.
[0157] The F98 glioma was injected in the striatum by stereotaxy
(day 0), and 6 days latter the animals were treated by
intracerebral injection of 60 .mu.L of 5 mM NFL-TBS.sub.40-63
peptide (or vehicle). At days 16, 24 or 30, rats were euthanized
and serial coronal sections of the brain were analyzed to detect
the NFL-TBS.sub.40-63 peptide and the glioma cells using anti-GFAP.
Cell nuclei were also counterstained by DAPI.
[0158] As shown in the FIG. 9, the NFL-TBS.sub.40-63 peptide (green
fluorescence) was detected in glioma cells at each time point and
not in the healthy surroundings cells (the peptide colocalizes with
GFAP staining).
[0159] Importantly, when non tumor-bearing (control) rats were
treated according to the same procedure, the NFL-TBS.sub.40-63
peptide was rapidly eliminated and was not detectable at these time
points. Moreover, no major cellular defects could be detected or
associated to the presence of the peptide when injected in normal
brain. In a physiological point of view, no clinical signs of
distress, such as weight loss or hunched posture were noticed when
these non tumor-bearing (control) rats were treated with the
NFL-TBS.sub.40-63 peptide.
[0160] All together these data indicate that the NFL-TBS.sub.40-63
peptide penetrates specifically in glioma cells in vivo and avoids
healthy cells. This is of particular interest, since this leads to
the conclusion that the NFL-TBS.sub.40-63 peptide has far less
toxic side effects than the other microtubule-targeting drugs (e.g.
paclitaxel, Cavaletti et al, 1997). To strengthen this, it has been
shown that the NFL-TBS.sub.40-63 peptide is indeed rapidly
eliminated when it is injected in a normal brain, favoring a poor
toxicity. This explains why, contrary to the other
microtubule-targeting drugs, the microtubule-targeting
NFL-TBS.sub.40-63 peptide does not induce any apparent effect when
injected in non tumor-bearing (control) rats.
3.3. Intracerebral Administration of NFL-TBS.sub.40-63 Reduces
Tumor Growth in Vivo
[0161] To further test whether the NFL-TBS.sub.40-63 peptide could
also inhibit the growth of glioma implanted in rat, serial sections
were stained with HE and the size of the tumor was evaluated by
morphometry using Image) (see an example of coronal section on FIG.
10A).
[0162] The animals treated with a single injection of the
NFL-TBS.sub.40-63 peptide exhibited significantly smaller tumors
than those observed in untreated animals. As a matter of fact, the
animals treated by the peptide exhibited a 71.7%.+-.18.9 reduction
of tumor volume (compared to vehicle treated animals) at day 16, a
72.0%.+-.21.2 reduction at day 24, and a 42.8%.+-.11.3 reduction at
day 30 (FIG. 10B).
[0163] A similar tumor inhibition was also observed using MRI
analysis on the same group of animals. The volume of this tumor is
reduced in NFL-TBS.sub.40-63 peptide-treated animals when compared
to un-treated animals (not shown).
[0164] Rats were also closely monitored for their weight loss, an
indirect indicator of tumor growth reflecting the therapeutic
effect of the injected agent. Daily weighing of animals showed that
the weight loss of NFL-TBS.sub.40-63 treated animals was
significantly lower when compared to untreated animals (FIG.
11).
[0165] All together, the above-presented results highlight the
significant capacity of the NFL-TBS.sub.40-63 peptide to:
[0166] a) penetrate in glioma cells in vivo, when injected
intratumorally,
[0167] b) induce apoptosis of the glioma cells in which it
penetrates,
[0168] c) avoid affecting the other non-tumoral regions of the
brain,
[0169] d) inhibit the progression of glioma in vivo, by mean of
only one injection.
3.4. Use of NFL-TBS2 Peptide for Targeting Nanocapsules to Glioma
Cells
[0170] Lipid nanocapsules containing a lipophilic flurorochrome
were obtained and coupled or not with the NFL-TBS2 peptide. Three
glioma cell lines (GL261, T98G and U87-MG cells) were treated for 1
or 6 hour(s) with different dilution of LNC containing the
lipophilic fluorochrome (DiD) coupled or not with the NFL-TBS2
peptide. Improved targeting of the glioma cell lines with the
NFL-TBS2-coupled LNC was observed by FACS (FIG. 14A), and by
confocal microscopy on living cells (FIG. 14B) or fixed cells (FIG.
14C).
[0171] In vivo experiments have also been performed in C57B1/6 mice
bearing a GL261 tumor. To detect the tumor, serial sections of the
brain were labelled with DAPI or using an antibody against GFAP
(Glial Fibrillary Acid Protein), the intermediate filament highly
expressed in glial tumors like glioblastoma. Importantly, when LNC
(DiD)-NFL-TBS2 are administered intracerebrally by stereotaxic
injection in the tumor-bearing mice, the LNC are able to reach the
glioma cells (see on the right of the image on FIG. 14D) and they
remain sequestered in the tumor tissue (they are not observed in
the healthy tissue).
[0172] These results thus allow considering the NFL-TBS.sub.40-63
peptide as a very promising tool for treating glioma tumor, either
in animals or in human beings. Besides its therapeutic efficiency
in reducing tumor size, the NFL-TBS.sub.40-63 peptide does not show
the strong neurotoxicity usually associated with this kind of
(microtubule-targeting) drugs. It is therefore embodied as being
the future therapeutic agent for treating patients suffering from
glioma.
BIBLIOGRAPHIC REFERENCES
[0173] Barth, R F (1998) J. Neurooncol 36: 91-102
[0174] Bocquet A, et al (2009) Neurosci 29: 11043-11054.
[0175] Budman D. R. (1997). Cancer Invest 15: 475-490.
[0176] Cavaletti G, et al (1997). Neurotoxicology 18: 137-145.
[0177] Dumontet C., (1999). J Clin Oncol 17: 1061-1070
[0178] Gottesman M M and Pastan I (1993). Annu Rev Biochem 62:
385-427.
[0179] Heurtault B., et al., Pharm. Res. 19 (2002), pp. 875-880
[0180] Hofer S and Herrmann R (2001). J Cancer Res Clin Oncol 127:
91-95.
[0181] Kaech S. and Banker G. (2006) Nat Protoc. 2006;
1(5):2406-15
[0182] McCarthy K D and de Vellis J (1980). J Cell Biol 85:
890-902.
[0183] Mollinedo F. et al., (2003). Apoptosis 8: 413-450.
[0184] Ray J, et al. (1993) PNAS USA. April 15; 90(8):3602-6.
[0185] Redgate et al. (1991). Laboratory animal science 41:
269-73.
[0186] Rocchetti et al (2007). European Journal of Cancer 2007, vol
43, n.degree. 12, p 1862-1868
[0187] Schrijvers D. et al. (1998). Curr Opin Oncol 10:
233-241.
[0188] Wang T H, et al. (2000). Cancer 88:2619-2628.
Sequence CWU 1
1
3124PRTartificialTBS motif of the human light chain neurofilament
protein, amino acids 1Tyr Ser Ser Tyr Ser Ala Pro Val Ser Ser Ser
Leu Ser Val Arg Arg 1 5 10 15 Ser Tyr Ser Ser Ser Ser Gly Ser 20
224PRTartificialartificial scrambled peptide (NFL-SCR) 2Ser Leu Gly
Ser Pro Ser Ser Ser Val Arg Ala Ser Tyr Ser Ser Ser 1 5 10 15 Arg
Ser Tyr Val Tyr Ser Ser Ser 20 320PRTartificialTBS motif of the
quail light chain neurofilament protein 3Tyr Ser Ser Ser Ala Pro
Val Ser Ser Ser Val Arg Arg Ser Tyr Ser 1 5 10 15 Ser Ser Gly Ser
20
* * * * *
References