Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
  • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
  • A61K49/0039—Coumarin dyes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
  • A61K49/0013—Luminescence
  • A61K49/0017—Fluorescence in vivo
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0056—Peptides, proteins, polyamino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers

Definitions

• Malignant gliomas are the most prevalent type of primary tumors of the central nervous system (CNS).
• CNS central nervous system
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• anti-tumor drugs that is able to treat gliomas, e.g. glioblastomas, without triggering neurotoxicity.
• 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).
• 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, LSI 87, 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.40-63”) is able to inhibit the proliferation of neuroblastoma and glioblastoma cell lines in vitro.
• TBS tubulin-binding site
• 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.
• 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.
• the present invention relates to an isolated amino acid sequence comprising the NFL-TBS 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).
• SEQ ID NO 1 a biologically active derivative thereof
• the present invention relates to the use of an amino acid sequence comprising the NFL-TBS 40 -63 peptide (SEQ ID NO 1), or a biologically active derivative thereof, for detecting glioma cells either in vivo, or in vitro.
• said method is a method for testing in vitro a biological sample for the presence or absence of malignant glioma cells, said method comprising: a. Suspending the cells of the sample in an appropriate medium, b. Mixing an amino acid sequence comprising the NFL-TBS 40 -63 peptide or a biologically active derivative thereof with the suspended cells of the sample, 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.
• said amino acid sequence is the NFL-TBS 40 -63 peptide (SEQ ID NO 1) itself.
• Figure 1 demonstrates the in vitro specificity of the penetration of the NFL-TBS 40 -63 peptide (10 ⁇ , 6h) in rat glioma cells (F98 and 9L), as compared to rat primary astrocytes and neurons, analyzed by immunohistochemistry (A).
• Cellular uptake of different doses of the NFL-TBS 40 -63 peptide (1, 5, 10, 20, 50, 100 ⁇ , lh, 37°C) is further analyzed by flow cytometry (B).
• Figure 2 demonstrates the in vitro specificity of the penetration of the NFL-TBS 40 -63 peptide (10 ⁇ , 6h) in human glioma cells (U87-MG and T98G) as compared to normal human astrocytes, analyzed either by immunohistochemistry (A).
• Cellular uptake of different doses of the NFL-TBS 40 -63 peptide (1, 5, 10, 20, 50, 100 ⁇ , lh, 37°C) is further analyzed by flow cytometry (B).
• Figure 3 shows the in vitro specificity of the penetration of the NFL-TBS 40 -63 peptide (10 ⁇ , 6h) in mouse glioma cells (GL261) as compared to mouse astrocytes, analyzed either by immunohistochemistry (A).
• Cellular uptake of different doses of the NFL- TBS 40 -63 peptide (1, 5, 10, 20, 50, 100 ⁇ , lh, 37°C) is further analyzed by flow cytometry (B).
• Figure 4 shows the in vitro survival of rat glioma cells (F98 and 9L) and rat primary astrocytes in the presence of various concentration of NFL-TBS 40 -63 peptide during 72 h assessed by the MTS assay (A).
• the microtubule cytoskeleton is completely disorganized in glioma cells but not in rat astrocytes and neurons, as assessed by immunohistochemistry (B).
• Figure 5 shows 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 40 -63 peptide during 72 h, assessed by the MTS assay (A).
• the microtubule cytoskeleton is completely disorganized in glioma cells but not in human astrocytes, as assessed by immunohistochemistry (B).
• Figure 6 shows the in vitro survival of the mouse glioma cells (GL261) and the mouse primary astrocytes in the presence of various concentration of NFL-TBS 40 -63 peptide during 72 h, assessed by the MTS assay (A).
• the microtubule cytoskeleton is completely disorganized in glioma cells but not in mouse astrocytes, as assessed by immunohistochemistry (B).
• Figure 7 shows the in vitro anti-proliferative activity of NFL-TBS 40 -63 peptide (100 ⁇ , 72h) as compared with taxol (40nM, 72h), NFL-SCR (100 ⁇ , 72h) on rat cells (F98, 9L, astrocytes, A), human cells (U87, T98G, astrocytes, B), and mouse cells (GL261, astrocytes, C).
• Figure 8 reveals that the NFL-TBS 40 -63 peptide (100 ⁇ , 72h) induces the in vitro apoptosis of rat glioma cells (A), of human and mouse glioma cells (B), but not of the corresponding astrocytes.
• Figure 9 shows that the injected NFL-TBS 40 -63 peptide (5mM/ 60 ⁇ ) 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 (A), 24 (B), or 30(C).
• Figure 10 shows the in vivo anti-proliferative effect of only one injection of the NFL- TBS 40 -63 peptide (5mM / 60 ⁇ ) on the growth of pre-implanted glioma in rat brain at days 16, 24, and 30 on coronal sections (A). Quantification of the tumor volume calculated from the coronal sections in peptide-treated or control animals at days 16, 24 and 30 (B).
• Figure 11 demonstrates the therapeutical activity of only one injection of the NFL- TBS 40 -63 peptide (5mM / 60 ⁇ ) by measuring the weight of the animals suffering from a glioma.
• Figure 12 shows the in vitro survival of primary human glioblastoma cells isolated after surgery in the presence of 100 ⁇ NFL-TBS 40 -63 peptide in comparison with 100 ⁇ NFL-SCR peptide or 40 nM taxol during 72 h assessed by the MTS assay.
• Figure 13 shows that uptake of NFL-TBS 40 -63 peptide is temperature and energy- dependant (A), (a) and (c): Glioma cells were incubated for 30 minutes at 37°C in the presence of 20 ⁇ fluorescein-tagged NFL-TBS 40 -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°C (black columns) or 4°C (white columns) in the presence of 20 ⁇ fluorescein-tagged NFL-TBS 40 -63 peptide.
• Figure 14 shows that NFL-TBS2 peptide can be used to improve the targeted uptake of lipid nanocapsules (LNC) in glioma cells.
• LNC lipid nanocapsules
• GL261 cells were treated for 6 hours, and U87-MG cells for 1 or 6 hours with different dilution of LNC containing a lipophilic fluorochrome (DiD). Cell fluorescence was measured by FACS (A). Images of living GL261 cells show higher fluorescence in cells treated with l0 ⁇ L ⁇ of LNC (DiD) coupled to NFL-TBS2 peptide for 6 hours than those treated with LNC (DiD) alone (B).
• LiD lipid nanocapsules
• 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) (D).
• 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.
• the present invention discloses the surprising selectivity of the microtubule- depolymerizing peptide NFL-TBS 4 0-63, 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).
• the NFL-TBS 4 0-63 peptide is 24 amino acids long and has the following sequence: YSSYSAPVSSSLSVRRSYSSSSGS (SEQ ID NO: l). 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).
• 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.
• 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.
• 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.
• the present invention provides an isolated amino acid sequence comprising the NFL-TBS 40 -63 peptide (SEQ ID NO: l), or a biologically active derivative thereof, for use in a method for treating malignant gliomas. 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 40 -63 (SEQ ID NO 1)) or a biologically active derivative thereof for the manufacture of a pharmaceutical composition for treating malignant glioma.
• the isolated amino acid sequence of the invention yet comprises the NFL-TBS 40 -
• the isolated amino acid sequence of the invention yet comprises the NFL-TBS 40 -63 peptide provided that it is not the entire neurofilament light (NFL) subunit itself.
• the isolated amino acid sequence comprises no more than 100 amino acids, preferably 50 amino acids.
• the isolated amino acid sequence of the invention consists of the NFL-TBS 40 -63 peptide (SEQ ID NO 1) or a biologically active derivative thereof. Preferably, it consists of the NFL-TBS 40 -63 peptide itself.
• the present invention makes use of the "biologically active derivative of the NFL- TBS 40 -63 peptide".
• 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 40 -63 (SEQ ID NO 1)) include variants and fragments of the NFL-TBS 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 40 -63 peptide.
• the "biologically active" derivatives of the NFL-TBS 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 40 -63 peptide.
• the antiproliferative effect of the derivatives of the NFL-TBS 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 40 -63 peptide, as assessed in vitro by conventional proliferation techniques.
• the derivatives of the NFL-TBS40-63 peptide have preferably the same specificity as the parent NFL-TBS40-63 peptide toward glioma cells, as assessed in vitro by conventional cellular uptake experiments.
• the derivative of the NFL-TBS 40 -63 peptide is a biologically active fragment of the NFL-TBS 40 -63 peptide.
• Said fragment comprises at least 12 successive amino acids of the parent NFL-TBS 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 40 -63 peptide.
• the derivative of the NFL-TBS 40 -63 peptide is a biologically active variant of the NFL-TBS 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 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 40 -63 peptide.
• such allelic variant has 70%, preferably 80%, more preferably 90% and even more preferably 95% of identity as compared with the parent NFL-TBS 40 -63 peptide (SEQ ID NOl).
• 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 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 stereo specificity 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 40 -63 peptide. All of these variations are well known in the art. Thus, given the peptide sequences of the NFL- TBS 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 40 -63 peptide retain at least the biological activity and specificity of the NFL-TBS 40 -63 peptide.
• the peptides used in the invention can be conveniently synthesized using art recognized techniques.
• 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/bl2.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).
• 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 40 -63 (SEQ ID NO 1) or a biologically active derivative thereof.
• 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.
• AA anaplastic astrocytoma
• GBM glioblastoma multiform
• AO anaplastic oligodendroglioma
• AOA anaplastic oligoastrocytoma
• GBM glioblastoma multiform
• said subject is a mammal, preferably a mouse, a rat, a cat, or a dog, and more preferably a human being.
• Such pharmaceutical composition comprises an isolated amino acid sequence comprising the NFL-TBS 40 -63 (SEQ ID NO 1) or a biologically active derivative thereof and a pharmaceutically acceptable carrier.
• the isolated amino acid sequence to be incorporated in the pharmaceutical composition of the invention is the NFL-TBS 40 -63 peptide (SEQ ID NO 1) or a biologically active derivative thereof, and, preferably, the NFL-TBS40-63 peptide itself.
• the peptide in another embodiment, 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 advantage 3.4. below).
• a radioactive moiety such as a cytotoxic component
• an appropriate carrier such as lipid nanocapsules as shown in advantage 3.4. below.
• suitable pharmaceutically acceptable carriers include, but are not limited to: water, salt solutions (e.g., NaCI), 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.
• 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.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• 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.
• 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.
• 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 fc 2 and C T values as described by Rocchetti et al (2007).
• the effective doses for treating animals range between about 0,1 micromole and 5 milimole using a single stereotaxic injection (60 ⁇ 1), 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.
• the NFL-TBS 4 0-63 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 40 -63 peptide in a specific manner, and can be identified among other healthy brain cells (in particular astrocytes and neurons).
• the NFL-TBS 4 0-63 peptide is thus a promising tool to detect glioma cells, either in vitro, or in vivo.
• the present invention relates to the use of an amino acid sequence comprising the NFL-TBS 40 -63 peptide (SEQ ID NO 1) or a biologically active derivative thereof for detecting glioma cells, preferably glioblastoma cells.
• 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.
• labeled 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 32 P, 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).
• FRET fluorescence resonance energy transfer
• 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.
• said labels are fluorochromes.
• Suitable fluorochromes include, for example:
• fluorescein and derivatives like hexachloro-fluorescein, tetrachloro- fluorescein, carboxyfluorescein (TAMRA), CAL FLUOR® (CAL Fluor
• succinimidyl ester of carboxyfluorescein succinimidyl ester of 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEXTM) or succinimidyl ester of 6-carboxy-4',5'-dichloro- 2' ,7 ' dimethoxyfluorescein (JOETM)
• HEXTM succinimidyl ester of 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein
• JETM succinimidyl ester of 6-carboxy-4',5'-dichloro- 2' ,7 ' dimethoxyfluorescein
• Rhodamine and derivatives like 5- or 6-carboxy-X-rhodamine (ROX), N,N,N',N'-tetramethyl-6-carboxyrhodamine; 3. Cyanine and derivatives like Cy3, Cy3.5, Cy5, Cy5.5;
• said fluorochrome is selected in the group comprising fluorescein and derivatives like hexachloro-fluorescein, tetrachloro-fluorescein, carboxyfluorescein (TAMRA), CAL FLUOR® (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 (HEXTM) or succinimidyl ester of 6-carboxy-4',5'-dichloro-2',7'dimethoxyfluorescein (JOETM)).
• TAMRA carboxyfluorescein
• CAL FLUOR® CAL Fluor Green 520, CAL FLUOR Gold
• preferred conventional techniques to detect such fluorochrome-labeled amino acid sequence include, but are not limited to, flow cytometry or fluorescence microscopy.
• the amino acid sequence of the invention that is used to detect glioma cells consists of the NFL-TBS 40 -63 peptide (SEQ ID NO 1) or a biologically active derivative thereof.
• the amino acid sequence of the invention is the NFL-TBS 40 -63 peptide itself, which is coupled for example with a carboxyfluorescein dye or a biotin tag.
• the present invention relates to the use of the isolated amino acid sequence comprising the NFL-TBS 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 40 -63 peptide or a biologically active derivative thereof, for its use for detecting the glioma cells in vivo.
• glioma cells are generally not located in a confined area because they are able to infiltrate the surrounding regions of the original tumor.
• 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.
• 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.
• 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.
• the present invention provides a method for in vivo detecting the presence of malignant glioma cells, said method comprising: a) labeling an amino acid sequence comprising the NFL-TBS 40 -63 peptide or a biologically active derivative thereof with a fluorescent or luminescent dye, directly or through an appropriate carrier (such as nanocapsules), b) injecting said amino acid sequence intracerebrally prior to a surgical operation, 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.
• the present invention is related to the use of the isolated amino acid sequence comprising the NFL-TBS 4 0-63 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.
• 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: 1. Suspending the cells of the sample in an appropriate medium,
• biological sample designates a cell culture that is handled in vitro.
• the cells in culture can be either of lineage origin or of primary origin.
• the cells can be extracted from an animal brain following a biopsy or a surgical operation.
• the percentage of cells containing said amino acid sequence "corresponds to" the percentage of glioma cells in the sample.
• 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%.
• 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%.
• 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% C0 2 at 37°C.
• 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.
• the concentration of the amino acid sequence to be added is comprised between 1 and 200 ⁇ , and more preferably between 2 and 150 ⁇ , and even more preferably between 5 and 50 ⁇ .
• 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.
• 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).
• a dye or a conventional tag e.g. biotin
• appropriate carrier such as nanocapsules
• 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).
• 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.
• conventional techniques of indirect detection can be used (e.g. flow cytometry, immunohistochemistry, Western Blot, etc .).
• 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.
• the in vitro method of detection of glioma cells uses the NFL-TBS 4 0-63 peptide itself. More preferably, it uses a carboxyfluorescein-labelled NFL-TBS 4 0-63 peptide or a biotin-tagged NFL-TBS 4 0-63 peptide.
• the present invention relates to the use of the isolated amino acid sequence comprising the NFL-TBS 40 -63 peptide or a biologically active derivative thereof for addressing or targeting chemical compounds to glioma cells in vitro or in vivo.
• the present invention relates to the isolated amino acid sequence comprising the NFL-TBS 4 0-63 peptide or a biologically active derivative thereof, for its use for addressing or targeting chemical compounds to the glioma cells in vivo.
• 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.
• 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.
• appropriate carriers e.g. nanocapsules, liposomes, micelles, or any encapsulation mean that is known by the man skilled in the art
• Said chemical compounds can be pharmaceutical compounds and/or labeling markers such as fluorescent molecules.
• Said pharmaceutical compounds are preferably cytotoxic drugs, for example antimitotic drugs.
• the chemical compounds are more preferably encapsulated in lipid nanocapsules as described below.
• the in vitro and in vivo methods of targeting glioma cells use the NFL-TBS40-63 peptide itself.
• Biotinylated or carboxyfluorescein-labeled peptides corresponding to the labeled tubulin-binding site of NFL (NFL-TBS 40 -63, SEQ ID NO: l) 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.
• the biotin molecule is linked to the N-terminal end of the peptides.
• the carboxyfluorescein molecule is linked to the N-terminal end of the peptides.
• 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% C0 2 (37°C) until reaching 80-90% confluence.
• 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% C0 2 (37 °C).
• 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°C. Dissociated cells were plated on coverslips precoated with 5 ⁇ g/ml poly-l-lysine and 7 ⁇ g/ml collagen at densities of 2 x 10 5 /ml and incubated at 37°C with a 5% C02 atmosphere. Twenty-four hours later, the plating solution was replaced by B-27 neurobasal medium, and the second day cytosine arabinoside (20 ⁇ ) was added to eliminate non-neural cells. Experiments were performed 7 days after plating.
• 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).
• 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% C0 2 (37°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.
• 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 ) in MW96 and let grow at 37 °C in a humidified incubator gassed with 5% C0 2 . The peptides and/or drugs are added 48 hours after their plating.
• Lipid nanocapsules were performed as previously described (Heurtault et ah, 2002). Briefly, Solutol HS-15, Lipoid S75-3, sodium chloride, Labrafac CC and water were mixed by magnetic stirring (200 rpm) at room temperature leading to an oil/water emulsion. After heating, an interval of transparency at 70°C can be observed, and the inverted phase "water in oil” is obtained at 85-87°C. Three temperature cycles alternating from 60 to 87 °C were applied, then before the last decrease of temperature, the mixture was diluted with 12.5mL of cold water (close to 0°C) and stirred for 15 min. DiD was added just before the last dilution.
• peptides were evaluated by the MTS cytotoxicity assay and by counting directly the number of cells.
• MTS assay Promega
• 500 cells were seeded in 96-well plates, incubated at 37°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.
• 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.
• glioma cells or astrocytes were seeded in 35 mm dishes and cultured for 1 hour at 37°C in media containing fluorescein-labeled NFL-TBS 4 0-63 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°C.
• 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 ⁇ 4'6-diaminido-2-phenylindole (DAPI; Sigma) for 5 min, and washed twice with PBS. Coverslips were mounted with an antifading solution. 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.
• 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 x 10 4 cells/mL in DMEM was obtained. Animals were anesthetized by intraperitoneal injection of a mixture of ketamine 10% (0.8 ⁇ /g), and xylazine 2% (0.5 ⁇ /g).
• 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 ⁇ of DMEM alone or containing 500 tumor cells was injected at a flow rate of 2 ⁇ /min using a 10- ⁇ 1 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).
• CED Slow-infusion Convection-Enhanced Delivery procedure
• Frozen brains were serially sectioned using a Leica cryostat, and 20 ⁇ 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.
• the preparations were counterstained with 3 ⁇ 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.
• DAPI 3 ⁇ 4'6-diaminido-2-phenylindole
• MRI Magnetic resonance spectroscopy
• TR 2,000ms
• mean echo time (Tern) 31.7ms
• RARE factor 8
• FOV 3 x 3cm
• matrix 128 x 128 nine contiguous slices of lmm, eight acquisitions.
• NFL-TBS 40 -63 (10 ⁇ ) 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.
• FACS Fluorescent-activated cell sorter
• the peptide also penetrates specifically in primary human glioblastoma cells isolated after surgery (data not shown). All together, these in vitro results show importantly that the NFL-TBS 4 o_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 4 o-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).
• Rat glioma cells (F98 and 9L) and astrocytes were treated with the NFL-TBS40-63 peptide or its control scrambled sequence (NFL-SCR) at different concentrations (0, 20, 50 and 100 ⁇ ) 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.
• BrdU staining analysis demonstrated a cytostatic effect of the NFL- TBS. 40 - 63 peptide on glioma cells.
• Microtubule-binding drugs are known to arrest proliferation and to induce cell death by apoptosis.
• F98, 9L glioma cells or astrocytes cells were incubated with 100 ⁇ 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.
• PS membrane phospholipid phopsphatidylserine
• TBS 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).
• the NFL-TBS 40 -63 peptide is therefore able to induce the death of glioma cells by apoptosis but has no effect on normal healthy brain cells.
• 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 ⁇ ⁇ of 5 mM NFL-TBS 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 40 -63 peptide and the glioma cells using anti-GFAP. Cell nuclei were also counterstained by DAPI.
• the NFL-TBS 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).
• the NFL-TBS 40 -63 peptide was rapidly eliminated and was not detectable at these time points.
• no major cellular defects could be detected or associated to the presence of the peptide when injected in normal brain.
• 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-TBS40-63 peptide.
• the animals treated with a single injection of the NFL-TBS 40 -63 peptide exhibited significantly smaller tumors than those observed in untreated animals.
• 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 (figure 10B).
• 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 40 -63 treated animals was significantly lower when compared to untreated animals ( Figure 11). All together, the above-presented results highlight the significant capacity of the -TBS40-63 peptide to:
• 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.
• LiD lipophilic fluorochrome
• Improved targeting of the glioma cell lines with the NFL-TBS2 -coupled LNC was observed by FACS (figure 14 A), and by confocal microscopy on living cells (figure 14B) or fixed cells (figure 14C).
• the NFL-TBS 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 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.

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