WO2014059279A2 - Materials and methods useful to treat neuroblastomas and pheochromocytomas - Google Patents

Abstract

Methods for affecting one or more of: proliferation, migration and invasion of tumor cells are described. One method includes contacting the tumor cell with an effective amount of a composition such as tianeptine.

Description

TITLE

MATERIALS AND METHODS USEFUL TO TREAT NEUROBLASTOMAS AND PHEOCHROMOCYTOMAS

CROSS -REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application Ser. No. 61/713,520 filed October 13, 2012, the entire disclosure of which is expressly incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

[0002] The invention specifically pertains to treatments related to cancer, particularly treatments for neuroblastomas and pheochromocytomas.

BACKGROUND

[0003] Neuroblastoma is a major neural crest cancer linked to -15% of cancer-related childhood deaths and, in many cases, is resistant to surgical and conventional chemotherapy. Unfortunately, most current chemotherapies have highly variable outcomes and are accompanied by short- and long-term side effects. Conventional cancer intervention methods, such as surgery, radiation therapy and chemotherapy, are often ineffective against neuroblastoma. In addition, surgery and radiation therapy are not possible in patients with metastatic neuroblastomas that invade various body parts, including the brain. Further, neuroblastoma is resistant to conventional chemotherapy, especially with taxanes, the microtubule inhibitors used in the standard treatment of advanced cancers. Moreover, most chemotherapies cause severe acute and chronic side effects on the development and activity of the brain (e.g., taxane causes neurotoxicity, nephrotoxicity and hypersensitivity). While a combination of four different drugs (carboplatin, cyclophosphamide, doxorubicin, etoposide) has been used to treat childhood cancers, including neuroblastoma, there are drawbacks to such therapies. Carboplatin and cyclophosphamide cause inter-strand crosslinking between DNAs. Doxorubicin intercalates and damages DNA. Etoposide inhibits DNA topoisomerase II and causes DNA strand breaks. The detrimental actions of these drugs on tumor DNA slow down or stop tumor growth. All of those drugs, however, cause both acute and chronic side-effects on the brain, which are even more severe on the child's brain.

[0004] Pheochromocytoma is an endocrine tumor arising in the adrenal medulla and paraganglia. Pheochromocytoma causes hyper- secretion of catecholamines, such as

norepinephrine and epinephrine, resulting in development of hypertension, headache, palpitation, anxiety and sweating. Surgical debulking of pheochromocytoma mass is used to reduce catecholamine hypersecretion. Pre-operative and intra-operative pharmacological treatments are administered to prevent a sudden surge of blood pressure caused by excessive catecholamine secretion during surgery. Approximately 10% of pheochromocytomas become malignant with high mortality rates. Surgery in combination with chemotherapy using cyclophosphamide, vincristine, dacarbazine, anthracycline, etoposide, cisplatin and sunitinib are used for the treatment of metastatic pheochromocytoma. If patients have unresectable metastatic pheochromocytoma, I- metaiodobenzylguanidine (MIBG) and somatostatin receptor radionuclide are used as alternative treatment. However, the prognosis of the above treatments of malignant pheochromocytomas is still poor. Thus, it is in great demand to find a better strategy for the treatment of metastatic pheochromocytoma.

[0005] While there exists chemotherapy using microtubule inhibitors (e.g. , taxane) as treatment of advanced cancers, such taxane treatment suppresses microtubule dynamics during the prophase-to-metaphase transition and prevents proper alignment of chromatin at the mid-plane. This, in turn, activates a cyclin B/CDK1 complex-dependent checkpoint that delays cell cycle progression until chromatin is correctly aligned at the mid-plane. If the delay persists, cyclin B/CDK1 complex induces p53-dependent cell apoptosis to prevent aneuploidy.

[0006] In addition, taxane-resistant cancer cells, including malignant pheochromocytoma and neuroblastoma cells, escape the checkpoint by degrading the cyclin B/CDK1 complex and p53, contributing to the development of more aggressive tumors with aneuploidy or polyploidy.

[0007] Also, taxane treatment is accompanied with severe acute and chronic side effects such as neurotoxicity, nephrotoxicity and hypersensitivity. Thus, these obstacles have limited usage of microtubule inhibitors such as taxane for treatment of some cancers despite the fact that inhibition of microtubule dynamics is one of the most effective ways to prevent proliferation, migration and invasion of tumors.

[0008] In spite of considerable research into therapies to treat neuroblastoma and pheochromocytoma, they remain difficult to treat effectively and the mortality observed in patients indicates that improvements are needed in the diagnosis, treatment and prevention of

neuroblastoma and pheochromocytoma.

SUMMARY OF THE INVENTION

[0009] Described herein, at least in part, is the discovery that tianeptine suppresses neuroblastoma and pheochromocytoma and enhances neurogenesis.

[0010] Described herein are results showing that tianeptine causes apoptosis by increasing microtubule bundling in neuroblastoma and pheochromocytoma cells. In addition, tianeptine blocks growth of neuroblastoma and pheochromocytoma by inducing specific and rapid degradation of cytoplasmic dynein, a microtubule motor protein that arranges the mitotic spindles during mitosis. Tianeptine also inhibits microtubule dynamics using collapsin response mediator protein-2 (CRMP2), a microtubule stabilizer. These unique actions stop growth of and even kill pheochromocytoma and neuroblastoma cells that usually escape from the microtubule inhibitor, taxane, via a mitotic slippage. Tianeptine also promotes neurogenesis of embryonic cortical neurons at the apoptosis-inducing concentration. Without being bound by any particular theory, it is believed that tianeptine causes cell-cycle arrest and apoptosis of neuroblastoma and pheochromocytoma cells by a novel microtubule-based mechanism.

[0011] Also described herein are compositions of matter comprising tianeptine and at least one additional cancer therapeutic. In particular, there are provided compositions wherein the cancer therapeutic is selected from chemotherapeutic drugs, toxins, immunological response modifiers, enzymes and radioisotopes.

[0012] Also described herein are compositions wherein the cancer therapeutic is selected from: paclitaxel, docetaxel, carboplatin, cyclophosphamide, doxorubicin and etoposide. Also provided are compositions wherein the composition is capable of causing apoptosis of pheochromocytoma and neuroblastoma cells. Also provided are compositions wherein the composition is capable of affecting a cellular process such as: increasing microtubule bundling, increasing anti-lamin-Bl antibody levels, increasing apoptosis, inhibiting GTPase Ran, increasing protein degradation, increasing cytoplasmic dynein degradation, inhibiting collapsing response mediator protein-2, increasing susceptibility to taxanes.

[0013] In another aspect, there is provided herein a method for affecting one or more of: proliferation, migration and invasion of tumor cells, comprising contacting the tumor cell with an effective amount of a composition comprising tianeptine.

[0014] In certain embodiments, the cells are taxane-resistant cancer cells.

[0015] In certain embodiments, the tumor cells comprise malignant pheochromocytoma cells.

[0016] In certain embodiments, the tumor cells comprise malignant neuroblastoma cells.

[0017] In certain embodiments, the composition comprises tianeptine and at least one additional cancer therapeutic.

[0018] In certain embodiments, the additional cancer therapeutic is selected from one or more of: chemotherapeutic drug, toxin, immunological response modifier, enzyme and radioisotope

[0019] In certain embodiments, the additional cancer therapeutic is selected from one or more of: paclitaxel, docetaxel, carboplatin, cyclophosphamide, doxorubicin and etoposide.

[0020] In certain embodiments, the composition is capable of causing apoptosis of neuroblastoma cells.

[0021] In certain embodiments, the composition is capable of affecting a cellular process selected from the group consisting of: increasing microtubule bundling, increasing anti-lamin-Bl antibody levels, increasing apoptosis, inhibiting GTPase Ran, increasing protein degradation, increasing cytoplasmic dynein degradation, inhibiting collapsing response mediator protein-2, increasing susceptibility to taxanes. [0022] In another aspect, there is provided herein a method to affect at least one mammalian neuroblastoma cell, comprising contacting at least one mammalian neuroblastoma cell with at least an effective amount of tianeptine.

[0023] In certain embodiments, the at least one mammalian neuroblastoma cell is derived from an animal selected from the group consisting of: mouse, rat, guinea pig, rabbit, cat, dog, monkey and human.

[0024] In certain embodiments, the at least one mammalian neuroblastoma cell is cultured in vitro.

[0025] In certain embodiments, the at least one mammalian neuroblastoma cell is an animal model.

[0026] In certain embodiments, the at least one mammalian neuroblastoma cell is a human cell.

[0027] In another aspect, there is provided herein a method of ameliorating neuroblastoma in a mammal in need of such amelioration, comprising administering a composition comprising tianeptine.

[0028] In certain embodiments, the neuroblastoma is present in a body location selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

[0029] In certain embodiments, the neuroblastoma is present in the brain.

[0030] In another aspect, there is provided herein a method to affect at least one

neuroblastoma cell, comprising contacting tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof with at least one neuroblastoma cell.

[0031] In another aspect, there is provided herein a method of ameliorating neuroblastoma in a mammal in need of such amelioration, comprising administering a therapeutically-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to a mammal in need of neuroblastoma amelioration.

[0032] In certain embodiments, the methods further comprise administering a cancer therapeutic selected from the group consisting of: chemotherapeutic drug, toxin, immunological response modifier, enzyme and radioisotope.

[0033] In certain embodiments, the methods further comprise administering a cancer therapeutic selected from the group consisting of: paclitaxel, docetaxel, carboplatin,

cyclophosphamide, doxorubicin and etoposide.

[0034] In certain embodiments, the methods further comprise physical removal of neuroblastoma cells via a method selected from the group consisting of: surgery, aspiration, dissection, ablation and electromagnetic fluctuations.

[0035] In certain embodiments, the mammal is selected from the group consisting of: mouse, rat, guinea pig, rabbit, cat, dog, monkey and human.

[0036] In another aspect, there is provided herein a method to cause neuroblastoma cell apoptosis, comprising contacting an apoptosis-effective amount of tianeptine, or a

pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to at least one neuroblastoma cell.

[0037] In another aspect, there is provided herein a method of ameliorating taxane-resistant neuroblastoma in a mammal in need of such amelioration, comprising administering a therapeutically-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to a mammal in need of taxane-resistant neuroblastoma amelioration.

[0038] In certain embodiments, the taxane-resistant neuroblastoma is present in a body location selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

[0039] In certain embodiments, the taxane-resistant neuroblastoma is present in the brain.

[0040] In another aspect, there is provided herein a method of inhibiting proliferation of a cell comprising contacting the cell with an amount of (lR,2R,3S,4S)-N4-(3- aminocarbonylbicyclo[2.2. l]hept-5-ene-2-yl)-5-fluoro- -N2-[3-methyl-4-(4-methylpiperazin- 1- yl)]phenyl-2,4-pyrimidinediamine effective to inhibit its proliferation.

[0041] In another aspect, there is provided herein a method of inhibiting microtubule bundling in a cell, comprising contacting the cell with an amount of tianeptine effective to inhibit microtubule bundling.

[0042] In another aspect, there is provided herein a method to affect at least one mammalian pheochromocytoma cell, comprising contacting at least one mammalian pheochromocytoma cell with at least an effective amount of tianeptine.

[0043] In certain embodiments, the at least one mammalian pheochromocytoma cell is derived from an animal selected from the group consisting of: mouse, rat, guinea pig, rabbit, cat, dog, monkey and human.

[0044] In certain embodiments, the at least one mammalian pheochromocytoma cell is cultured in vitro.

[0045] In certain embodiments, the at least one mammalian pheochromocytoma cell is an animal model.

[0046] In certain embodiments, the at least one mammalian pheochromocytoma cell is a human cell.

[0047] In another aspect, there is provided herein a method of ameliorating

pheochromocytoma in a mammal in need of such amelioration, comprising administering a composition comprising tianeptine.

[0048] In certain embodiments, the pheochromocytoma is present in a body location selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

[0049] In certain embodiments, the pheochromocytoma is present in the brain [0050] In another aspect, there is provided herein a method to affect at least one

pheochromocytoma cell, comprising contacting tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof with at least one pheochromocytoma cell.

[0051] In another aspect, there is provided herein a method of ameliorating

pheochromocytoma in a mammal in need of such amelioration, comprising administering a therapeutically-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to a mammal in need of pheochromocytoma amelioration.

[0052] In certain embodiments, the methods further comprise physical removal of pheochromocytoma cells via a method selected from the group consisting of: surgery, aspiration, dissection, ablation and electromagnetic fluctuations.

[0053] In another aspect, there is provided herein a method to cause pheochromocytoma cell apoptosis, comprising contacting an apoptosis-effective amount of tianeptine, or a

pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to at least one pheochromocytoma cell.

[0054] In another aspect, there is provided herein a method of ameliorating taxane-resistant pheochromocytoma in a mammal in need of such amelioration, comprising administering a therapeutically-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to a mammal in need of taxane-resistant pheochromocytoma amelioration.

[0055] In certain embodiments, the taxane-resistant pheochromocytoma is present in a body location selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

[0056] In certain embodiments, the methods further comprise physical removal of taxane- resistant pheochromocytoma cells via a method selected from the group consisting of: surgery, aspiration, dissection, ablation and electromagnetic fluctuations.

[0057] In certain embodiments, the composition comprising tianeptine comprises predominantly the (R)-enantiomer of tianeptine and is substantially free of the (S)-enantiomer of tianeptine.

[0058] In certain embodiments, the composition comprising tianeptine comprises predominantly the (S)-enantiomer of tianeptine and is substantially free of the (R)-enantiomer of tianeptine.

[0059] In certain embodiments, the composition comprising tianeptine comprises a mixture of the (R)- and (S)-enantiomers of tianeptine. BRIEF DESCRIPTION OF THE FIGURES

[0060] The patent or application file may contain one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fees.

[0061] FIGS. 1A - IF: PC12 cells were treated with none (FIG. 1A), or ΙΟΟμΜ tianeptine for 12 hr (FIG. IB - FIG. IF) and immunostained using anti-a-tubulin antibody. Tianeptine- treated PC 12 cells lost interphase microtubules and accumulated bundled microtubules at the cell periphery. In addition, tianeptine-treated cells showed aberrant mitotic spindles, such as single (FIG. 1C) and multiple asters (FIG. ID), sparse metaphase spindles (FIG. IE) and incomplete abscission (FIG. IF).

[0062] FIG. 1G: Bar graph showing the average percent of cells with interphase and bundled microtubules and mitotic spindles. Three independent experiments (100 cells per experiment [Exp]) were performed to obtain the mean percent of cells and standard error of mean (SEM).

[0063] FIG. 1H: Cells were treated with ΙΟΟμΜ tianeptine twice at a 12-h interval. More severe bundling and aberration of microtubules were caused by double tianeptine treatment.

[0064] FIG. II: Cells were treated with 50μΜ paclitaxel (12 hr) and immunostained using anti- a -tubulin antibody. Paclitaxel caused tubulin aggregates throughout the cytoplasm. Scale bars = 5 μηι.

[0065] FIGS. 2A-2D: Untreated PC12 cells were incubated on ice for 0 (FIG. 2A), 10 (FIG. 2B), 20 (FIG. 2C) and 30 (FIG. 2D) min, fixed in -20°C methanol and immunostained using anti- α tubulin antibody. Most microtubules were depolymerized within 10 min after incubation on ice.

[0066] FIGS. 2E-2H: PC12 cells treated with 100 μΜ tianeptine for 18 hr were incubated on ice for 0 (FIG. 2E), 10 (FIG. 2F), 20 (FIG. 3G) and 30 (FIG. 2H) min, fixed in -20°C methanol and immunostained using anti-a-tubulin antibody. Even after 30-min incubation on ice, bundled microtubules were not completely depolymerized. Scale bars = 5 μηι.

[0067] FIG. 21: Bar graph showing the average intensities (+SEM) of immunostained microtubules at the cell periphery. The average intesities were quantified using Metamorph software. Those in control (untreated) cells were significantly decreased after 10-min incubation on ice. Compared to control (p<0.001, n=40 cells), the average microtubule intensities at the cell periphery were not decreased, but slightly increased, after 20-min incubation on ice and then decreased by -23%.

[0068] FIG. 2J: Microtubules were polymerized in the cell cytosols added with none, 20 μΜ paclitaxel, or 50 μΜ tianeptine. Polymerized microtubules were pelleted and free tubulins remained in the supernatant after ultracentrifugation. Tubulins in the pellet (P) and supernatant (S) were detected by immunoblotting.

[0069] FIG. 3A: CRMP2 and microtubules were immunostained by anti-CRMP2 and anti- a -tubulin antibodies. In untreated PC 12 cells, CRMP2 did not show significant colocalization with microtubules.

[0070] FIG. 3B: Tianeptine treatment (100 μΜ, 18 hr) increased colocalization between CRMP2 and aberrant mitotic spindles. Scale bars = 5μηι.

[0071] FIG. 3C: Bar graph showing average colocalization coefficients between CRMP2 and microtubules in cells treated with none (control) or 100 μΜ tianeptine were measured using Metamorph. Colocalization coefficients were significantly increased from 0.51+0.02 to 0.73+0.02 (mean +SEM, n=30 cells, p<0.0001).

[0072] FIG. 4A: Effects of different concentrations of tianeptine (100-400 μΜ, 18 hr) treatment on the protein levels of cytoplasmic dynein (DIC: dynein intermediate chain), dynactin (pl50), a-tubulin and kinesin-1 (KHC) in PC12 cells.

[0073] FIG. 4B: Line graph showing the densities of protein bands (FIG. 4A) of DIC, pl50, a -tubulin and KHC in the presence of different concentrations of tianeptine. DIC was more rapidly decreased than other proteins at 100 μΜ tianeptine. Dynactin, a-tubulin and kinesin-1 (KHC) were decreased by 2 folds at higher concentrations than 200 μΜ tianeptine.

[0074] FIGS. 4C and 4D: Immunostained cytoplasmic dynein in untreated cells (FIG. 4C) and in 100 μΜ (18 hr) tianeptine-treated cells (FIG. 4D). Tianeptine-treated cells showed much dimmer intensities of immunostained cytoplasmic dynein. Scale bars = 5 μηι.

[0075] FIG. 4E: Bar graph showing the average intensities (+SEM) of cytoplasmic dynein (DIC) immunostaining. Average intensities were quantified by Metamorph. The average intensities of tianeptine-treated cells (28.1+1.4) were -2.6 fold lower than those of control (72.6+2.5) (n=40 cells, p<0.001).

[0076] FIGS. 5A and 5B: Microtubules and dynactin in tianeptine-treated PC 12 cells were visualized using anti-a-tubulin and anti-pl50 antibodies. Cells treated once with 100 μΜ tianeptine showed association of dynactin with bundled microtubules (FIG. 5A). The extents of association (colocalization) of dynactin with microtubules were significantly increased after double tianeptine treatment at a 12-hr interval (FIG. 5B).

[0077] FIGS. 5C and 5D: PC12 cells transfected with GFP tag alone (FIG. 5C) or GFP- tagged dynamitin (FIG. 5D) were immunostained with anti-a-tubulin antibody. Compared to GFP-expressing cells, GFP-dynamitin-expressing cells showed microtubule bundling similarly to tianeptine-treated cells. Scale bars = 5 μηι.

[0078] FIGS. 6A - 6C: PC12 cells were treated for 18 hr with none (FIG. 6A), 50 μΜ paclitaxel (FIG. 6B), or 100 μΜ tianeptine (FIG. 6C) and their chromosomes in the nucleus were visualized with propidium iodide. Scale bars = 5 μηι. Paclitaxel (FIG. 6B) increased the number of cells showing fragmented chromosomes and polyploidy (marked '#') while tianeptine (FIG. 6C) did not. Tianeptine-treated cells showed abnormal chromosomes, nuclear collapse and inclusions (all marked '*'). Scale bars = 5 μηι. [0079] FIG. 6D: Bar graph showing the average percent (+SEM) of cells with polyploidy. Only paclitaxel-treated cells (48%+2) formed polyploidy (n=30 cells, p<0.001).

[0080] FIG. 6E: Bar graph showing the average (+SEM) of relative percents of live cells compared to control (untreated cells). The percent of live cells was increased by 1.96 fold in paclitaxel-treated cells (n=3 Exps, p<0.02) while decreased to -63% of control (n=3 Exps, p<0.05).

[0081] FIG. 6F: Bar graph showing the percents of apoptotic cells (BrdU-positive or nuclear collapse/inclusions) were calculated. Compared to control, tianeptine, but not paclitaxel, increased the percent of apoptotic cells by ~11.5 folds.

[0082] FIGS. 7A and 7B: PC12 cells treated with none (FIG. 7A) or 100 μΜ tianeptine (FIG. 7B) were permeabilized in 0.1% Tx-100 and immunostained with anti-lamin-B 1 antibody. Tianeptine treatment increased the percent of cells showing lamin-B 1 aggregation, an indicative of apoptosis. Scale bar = 5 μηι.

[0083] FIG. 7C: Bar graph showing the average percent (+SEM) of cells with lamin-B 1 aggregation. -75% of tianeptine-treated cells showed lamin-B 1 aggregation while none of control did (n>175, p<0.0001).

[0084] FIG. 7D: Effects of tianeptine and paclitaxel treatment on the protein levels of p53, 116kD PARP, caspase 3 and lamin-B 1. Compared to control, tianeptine treatment slightly increased p53 while paclitaxel-treated cells lost most of p53. 116kD PARP was also significantly decreased in tianeptine-treated cells but not in paclitaxel-treated cells. Caspase 3 and lamin-B 1 were decreased by neither tianeptine nor paclitaxel.

[0085] FIGS. 8A and 8B: The human neuroblastoma SH-SY5Y cells were treated with none (FIG. 8A) or ΙΟΟμΜ tianeptine for 18 hr (FIG. 8B) and immunostained using anti-a-tubulin antibody. Tianeptine-treated SH-SY5Y cells lost interphase microtubules and accumulated bundled microtubules at the cell periphery. In addition, tianeptine-treated cells showed aberrant mitotic spindles. Scale bars = 5 μηι.

[0086] FIG. 8C: Bar graph showing the average percent (+SEM) of cells showing bundled microtubules and aberrant mitotic spindles. 85%+7 of tianeptine-treated cells showed bundled microtubules and aberrant mitotic spindles compared to control (n=200, P<0.0001).

[0087] FIG. 8D: Effects of tianeptine treatment on the protein levels of DIC (dynein), p53, pi 50 (dynactin), 65kD PARP, KHC (kinesin-1), a- tubulin and lamin-B 1. Compared to control, tianeptine treatment caused complete loss of DIC while slightly increasing p53. pl50, 65kD PARP, KHC and a-tubulin were also significantly decreased by tianeptine treatment.

[0088] FIGS. 9A and 9B: E18 rat embryonic cortical neurons were treated with none (control, FIG. 9 A) or 100 μΜ tianeptine (FIG. 9B) for 18 hr and immunostained with anti-a- tubulin antibody (green) and phalloidin actin (red). Tianeptine-treated cells formed longer and more branched neurites. Scale bars = 10 μηι. [0089] FIG. 9C: Bar graph showing the average number (+SEM) of neurites in neurons. Tianeptine-treated neurons formed 3-fold more neurites than control (n= 15 neurons, p<0.0001).

[0090] FIG. 9D: Bar graph showing the average (+SEM) of total neurite length of each neuron. The total neurite length was increased by 3.7 fold in tianeptine-treated neurons (n= 15 neurons, p<0.0001).

[0091] FIG. 10: Pheochromocytoma PC-12 cells were treated twice at a 12-h interval with increasing concentrations of tianeptine and processed for protein extraction and immunoblotting. An immunoblot shows the protein levels of a-tubulin, cytoplasmic dynein, p53 and p27. a -tubulin was decreased slightly by treatment with increasing concentrations of tianeptine. However, cytoplasmic dynein was rapidly decreased to none even at 1 mM tianeptine. Two tumor suppressor proteins, p53 and p27, were gradually increased by treatment with increasing concentrations of tianeptine. The protein level of p27 reached to its peak at 10 mM tianeptine while that of p53 did so at 50 mM tianeptine.

DETAILED DESCRIPTION OF THE INVENTION

[0092] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

[0093] Terms

[0094] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided.

[0095] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9) and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0096] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia and is used herein to indicate a non- limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0097] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0098] Also, the use of "comprise," "contain," and "include," or modifications of those root words, for example but not limited to, "comprises," "contained," and "including," are not intended to be limiting. The term "and/or" means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, "X and/or Y" can mean "X" or "Y" or "X and Y".

[0099] The term "combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.

[00100] The terms "agent" and "drug" generally refer to any therapeutic agents (e.g., chemotherapeutic compounds and/or molecular therapeutic compounds), antisense therapies, radiation therapies, or surgical interventions, used in the treatment of a particular disease or disorder.

[00101] The term "adjunctive therapy" generally refers to a treatment used in combination with a primary treatment to improve the effects of the primary treatment.

[00102] The term "clinical outcome" generally refers to the health status of a subject following treatment for a disease or disorder, or in the absence of treatment. Clinical outcomes include, but are not limited to, an increase in the length of time until death, a decrease in the length of time until death, an increase in the chance of survival, an increase in the risk of death, survival, disease- free survival, chronic disease, metastasis, advanced or aggressive disease, disease recurrence, death and favorable or poor response to therapy.

[00103] The term "decrease in survival" generally refers to a decrease in the length of time before death of a subject, or an increase in the risk of death for the subject.

[00104] The term "control" generally refers to a sample or standard used for comparison with an experimental sample, such as a sample obtained from a subject. In some embodiments, the control is a sample obtained from a healthy subject. In some embodiments, the control is cell/tissue sample obtained from the same subject. In some embodiments, the control is a historical control or standard value (i.e., a previously tested control sample or group of samples that represent baseline or normal values, such as the level in a control sample). In other embodiments, the control is a sample obtained from a healthy subject, such as a donor. Test samples and control samples can be obtained according to any method known in the art. [00105] The terms "prevent," "preventing" and "prevention" generally refer to a decrease in the occurrence of disease or disorder in a subject. The prevention may be complete, e.g., the total absence of the disease or disorder in the subject. The prevention may also be partial, such that the occurrence of the disease or disorder in the subject is less than that which would have occurred without the present invention. "Preventing" a disease generally refers to inhibiting the full development of a disease.

[00106] The terms "treating" and/or "ameliorating a disease" generally refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or disorder after it has begun to develop. "Ameliorating" generally refers to the reduction in the number or severity of signs or symptoms of a disease or disorder.

[00107] The term "subject" includes human and non-human animals. The preferred subject for treatment is a human. "Subject" and "subject" are used interchangeably herein.

[00108] The term "therapeutic" generally is a generic term that includes both diagnosis and treatment.

[00109] The term "therapeutic agent" generally refers to a chemical compound, small molecule, or other composition, such as an antisense compound, protein, peptide, small molecule, nucleic acid, antibody, protease inhibitor, hormone, chemokine or cytokine, capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. "Incubating" includes a sufficient amount of time for an agent to interact with a cell or tissue. "Contacting" includes incubating an agent in solid or in liquid form with a cell or tissue. "Treating" a cell or tissue with an agent includes contacting or incubating the agent with the cell or tissue.

[00110] The term "therapeutically effective amount" generally refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disease or disorder. For example, a therapeutically effective amount will refer to the amount of a therapeutic agent that decreases the rate of rejection, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[00111] A "therapeutically effective amount" can be a quantity of a specified pharmaceutical or therapeutic agent sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent. For example, this can be the amount of a therapeutic agent that prevents, treats or ameliorates the disease or disorder in a subject. The effective amount of the agent will be dependent on several factors, including, but not limited to the subject or cells being treated and the manner of administration of the therapeutic composition.

[00112] The term "pharmaceutically acceptable vehicles" generally refers to such

pharmaceutically acceptable carriers (vehicles) as would be generally used. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 20 Edition, describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules or agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[00113] General Description

[00114] Various chemotherapies have been developed to treat surgically-inoperable childhood cancers including brain cancers. Efficacy is decreased by three major obstacles: drug resistance, severe acute/chronic side effects and impermeability of the blood-brain barrier (BBB). For example, usage of taxane and other microtubule inhibitors, the conventional therapeutic approaches for treatment of various advanced tumors, is limited in childhood cancers because they show all of the above problems, despite the fact that inhibition of microtubule dynamics is one of the most effective ways to prevent proliferation, migration and invasion of tumors.

[00115] It is desirable to discover and develop drugs and compounds that inhibit microtubule dynamics while avoiding the above described drawbacks to currently used microtubule inhibitors. Described herein is a use of tianeptine in the treatment and cure of neuroblastoma and pheochromocytoma. Shown now herein is the ability of tianeptine to inhibit microtubule dynamics and cause apoptosis in neuroblastoma and pheochromocytoma cells.

[00116] Tianeptine is a drug known for effectively alleviating the symptoms of mild-to-severe major depression. The anti-depressant activity of tianeptine is mediated by enhancement of serotonin reuptake into nerve terminals for recycling. Tianeptine also displays significant anxiolytic properties and is useful in treaty a variety of anxiety disorders.

[00117] Tianeptine is now shown herein to cause formation of bundled microtubules and aberrant mitotic spindles in PC12 cells. The bundled microtubules in tianeptine-treated cells are resistant to cold-induced depolymerization. It is also shown herein that tianeptine does not stabilize microtubules directly, but rather that tianeptine enhances association of the microtubule stabilizer, CRMP2, with microtubules, which contributes to formation of aberrant mitotic spindles. Furthermore, tianeptine rapidly decreases the protein level of cytoplasmic dynein and that dynactin disassembly, that inhibits cytoplasmic dynein, causes microtubule bundling. It is also shown herein that along with causing abnormal nuclei, tianeptine increases aggregation of lamin-Bl . Additionally, it is shown herein that tianeptine causes apoptotic protein degradation while stabilizing p53 in PC 12 cells, and causes microtubule bundling and specific degradation of cytoplasmic dynein in human neuroblastoma cells. It is also shown herein that tianeptine does not cause apoptosis in rat embryonic cortical neurons, but does cause specific degradation of cytoplasmic dynein and increases the protein levels of tumor supressors.

[00118] Tianeptine, which has the systematic name 7-[(3chloro-6,ll-dihydro-6-methyl- dibenzo[c,f][l,2]thiazepinll-yl)amino]heptanoic acid S,S-dioxide, is an antidepressant of the dibenzothiazepine type. The structure of tianeptine, given in Formula I indicates that the absolute conformation about the asymmetric carbon may be either (R) or (S).

Formula I

[00119] The (R) or (S) enantiomers of tianeptine may be isolated. Thus, in certain embodiments, the (R)-enantiomer of tianeptine which is substantially free of the corresponding (S)-enantiomer, or the (S)-enantiomer of tianeptine which is substantially free of the corresponding (R)-enantiomer, is used in the present methods.

[00120] The compounds of Formula I described above may take the form of a

pharmaceutically-acceptable salt. The term "salts," embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases.

[00121] For example, pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Suitable organic acids include aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, such as formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, beta-hydroxybutyric, galactaric and galacturonic acid.

[00122] Suitable pharmaceutically acceptable base addition salts of the compounds of Formula I include metallic salts made from calcium, magnesium, potassium, sodium and zinc, or organic salts made from Ν,Ν'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.

[00123] As used herein, an "effective amount" of a compound of Formula I used to treat neuroblastoma or pheochromocytoma refers to the amount of the compound that treats neuroblastoma or pheochromocytoma, or prevents or alleviates one or more symptoms of neuroblastoma or pheochromocytoma. Similarly, an "effective amount" of a compound of Formula I used to treat neuroblastoma or pheochromocytoma refers to the amount of the compound that prevents or alleviates the neuroblastoma or pheochromocytoma. Likewise, an "effective amount" of a compound of Formula I used refers to the amount of the compound that prevents or alleviates one or more symptoms. A physician can readily determine when symptoms are prevented or alleviated, for example through clinical observation of a subject, or through reporting of symptoms by the subject during the course of treatment.

[00124] One skilled in the art can readily determine an effective amount of a compound of Formula I to be administered, by taking into account factors such as the size, weight, age and sex of the subject, the extent of disease penetration or persistence and severity of symptoms and the route of administration. Generally, an effective amount of the compounds of Formula I administered to a subject is from about 2 to about 100 mg per day, preferably from about 5 to about 60 mg per day and more preferably about 30 mg per day. Higher or lower doses are also contemplated.

[00125] The compounds of Formula I may be administered to a subject by any route, for example by enteral (e.g., oral, rectal, intranasal, etc.) and parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intravaginal, intravesical (e.g., into the bladder), intradermal, topical or subcutaneous administration. Also contemplated is the instillation of the compounds of Formula I into the body of the subject, for example in a controlled release formulation, with systemic or local release of the compound to occur over time or at a later time. According to some preferred embodiments, the compound of Formula I is localized in a depot for controlled release to the circulation or to a local site such as the gastrointestinal tract.

[00126] In the practice of the present methods, compounds of Formula I may be administered in the form of a pharmaceutical composition comprising at least one compound of Formula I and a pharmaceutically acceptable carrier. Pharmaceutical formulations may comprise from 0.1 to 99.99 weight percent of at least one compound of Formula I. Pharmaceutical compositions may be formulated according to standard practices in the field of pharmaceutical preparations. See, Alphonso Gennaro, ed., Remington 's Pharmaceutical Sciences, 18th Ed., (1990) Mack Publishing Co., Easton, Pa. Suitable dosage forms may comprise, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.

[00127] By "pharmaceutically acceptable carrier" is meant any diluent or excipient that is compatible with the other ingredients of the formulation and which is not deleterious to the recipient. The pharmaceutically acceptable carrier may be selected on the basis of the desired route of administration, in accordance with standard pharmaceutical practices.

[00128] Described herein are pharmaceutical compositions for parenteral administration may take the form of an aqueous or nonaqueous solution, dispersion, suspension or emulsion. In preparing pharmaceutical compositions for parenteral administration, at least one compound of Formula I may be mixed with a suitable pharmaceutically acceptable carrier such as water, oil (particularly a vegetable oil), ethanol, saline solutions (e.g., normal saline), aqueous dextrose (glucose) and related sugar solutions, glycerol, or glycols such as propylene glycol or polyethylene glycol. Pharmaceutical compositions for parenteral administration preferably contain a water- soluble salt of at least one compound of Formula I. Stabilizing agents, antioxidant agents and preservatives may also be added to the pharmaceutical compositions for parenteral administration. Suitable antioxidant agents include sulfite, ascorbic acid, citric acid or salts thereof and ethylenediaminetetraacetic acid (EDTA) or a salt thereof. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorbutanol.

[00129] In preparing pharmaceutical compositions for oral administration, at least one compound of Formula I may be combined with one or more solid or liquid inactive ingredients to form tablets, capsules, pills, powders, granules or other suitable oral dosage forms. For example, at least one compound of Formula I may be combined with at least one pharmaceutically acceptable carrier such as a solvent, filler, binder, humectant, disintegrating agent, solution retarder, absorption accelerator, wetting agent absorbent or lubricating agent. In one embodiment, at least one compound of Formula I is combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch and is formed into tablets by conventional tableting methods.

[00130] Pharmaceutical compositions as described herein may also be formulated so as to provide controlled-release of at least one compound of Formula I upon administration of the composition to a subject. Preferably, a controlled-release pharmaceutical composition is capable of releasing at least one compound of Formula I into a subject at a desired rate, so as to maintain a substantially constant pharmacological activity for a given period of time.

[00131] Formulation of controlled-release pharmaceutical compositions is within the skill in the art. Controlled release formulations suitable for use are described in, for example, U.S. Pat. Nos. 5,674,533 (liquid dosage forms), U.S. Pat. No. 5,059, 595 (gastro-resistant tablet), U.S. Pat. No. 5,591,767 (liquid reservoir transdermal patch), U.S. Pat. No. 5, 120,548 (device comprising swellable polymers), U.S. Pat. No. 5,073,543 (ganglioside-liposome vehicle), U.S. Pat. No. 5,639,476 (stable solid formulation coated with a hydrophobic acrylic polymer), the entire disclosures of which are herein incorporated by reference.

[00132] Biodegradable microparticles may also be used to formulate controlled-release pharmaceutical compositions suitable for use, for example as described in U.S. Pat. Nos. 5,354,566 and 5,733,566, the entire disclosures of which are herein incorporated by reference.

[00133] In one embodiment, controlled-release pharmaceutical compositions comprise at least one compound of Formula I and a controlled-release component. As used herein, a "controlled- release component" is a compound such as a polymer, polymer matrix, gel, permeable membrane, liposome and/or microsphere that induces the controlled-release of the compound of Formula I into the subject upon exposure to a certain physiological compound or condition. For example, the controlled-release component may be biodegradable, activated by exposure to a certain pH or temperature, by exposure to an aqueous environment, or by exposure to enzymes.

[00134] In one embodiment, compositions comprising at least compound of Formula I, or an analogue, metabolite, precursor, salt or formulation thereof are contacted with at least one neuroblastoma cell, resulting in apoptosis. As used herein an "apoptosis-effective amount" of a compound of Formula I refers to the amount of the compound that induces apoptosis in a neuroblastoma cell. One skilled in the art can readily determine an effective amount of a compound of Formula I to be administered in order to cause apoptosis of a neuroblastoma cell or a pheochromocytoma cell.

[00135] Examples

[00136] Certain embodiments of the present invention are defined in the Examples herein. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

[00137] Example 1

[00138] Materials and Methods

[00139] Cell culture and drug treatment

[00140] PC12 (rat pheochromocytoma) and SH-SY5Y (human neuroblastoma) cells were grown in Dulbecco's modified Eagle' s medium (DMEM) (GIBCO-BRL, Grand Island, NY) containing 10% FBS (GIBCO-BRL), 5% horse serum (GIBCO-BRL: omitted for culturing of SH- SY5Y cells) and 1 x Pen Strep (Invitrogen, Carlsbad, CA). 2 x 10⁴ cells were seeded on 25 x 25 mm coverslips in 35 mm dishes or 6-well plates and grown for 18 hr in growth medium before subjecting to transfection and immunocytochemistry procedures. Transfection of DNA constructs was performed using Lipofectamine™ 2000 according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). DNA (4 μg) plus lipofectamine (10 μΐ) in OPTI-MEM I (GIBCO-BRL) and 1.5 ml DMEM medium were added to the cells, followed by incubation for 18 hr. El 8 rat cortical neurons were purchased from GenLantis Inc. (San Diego, CA) and incubated in 10% FBS plus DMEM for 2 days for recovery and in B27 neurobasal medium (Genlantis, San Diego, CA) for 14 days for differentiation at 37°C with 5% C0₂/95 air. For drug treatment, PC12 or SHSY5Y cells in DMEM plus 10% FBS were treated with tianeptine (100-400 μΜ), paclitaxel (50 μΜ), or water for 12-24 hr.

[00141] Immunocytochemistry

[00142] For immunocytochemistry, all steps were performed at room temperature unless otherwise noted. Cells were cultured on glass coverslips coated with 0.001 poly-Lysine (Sigma- Aldrich, St. Louis, MO). Cells were rinsed with PBS, fixed in 3.5% formaldehyde in PBS for 30 min and permeabilized in 0.1 % Triton X-100 in PBS for 30 min. Cells were then blocked in TBS containing 0.1 % Tween-20 and 2% BSA and incubated for 30 min in primary antibodies. Primary antibodies to a-tubulin, kinesin-1 (KHC), cytoplasmic dynein (DIC), lamin-B l, or dynactin (pl50) were purchased from Santa Crz (Santa Cruz, CA) and those to PARP, caspase 3, CRMP-2 from Abeam (Cambridge, MA). Bound primary antibodies were detected using their corresponding secondary antibodies conjugated with Alexa₄₈8nm or Alexa_594nm (Invitrogen). Coverslips were then mounted on glass slides using Fluoromount G (Fisher, Hanover Park, IL).

[00143] Confocal microscopy and image analysis

[00144] The images of immunostained PC 12 and SH-SY5 Y cells were taken using a TCS SP5 multi-photon laser scanning confocal microscope (Leica Microsystems, Bamiockbum, IL) equipped with conventional solid slate, a ti-sapphire tunable multi-photon laser (Coherent, Santa Clara, CA) and acousto optical beam splitter AOBS. Images were acquired with 63 x Zeiss alpha plan fluor oil objective (1.4 NA) using the sequential scan mode to eliminate any spectral overlap in the individual fluorophores. Images were digitized using 'Leica Microsystems' software. The Metamorph software (Molecular Devices, Downingtown, PA) was used to quantify

immunostaining intensities and calculate colocalization coefficient. Region of Interest was limited to one cell per image.

[00145] Immunoblotting and protein analysis

[00146] One 6-well plate at about 90% confluency was incubated in 10% FBS-containing medium and treated with drugs. After drug treatment, cells in each well were harvested with 0.05% Trypsin-EDTA and washed once in PBS. The cells were then resuspended in an equal volume of ice-cold homogenizing PMEE buffer (35 mM Pipes, 35 mM KOH, 5 mM MgS04, 1 mM EGTA, 0.5 mM EDTA and 1 mM DTT) plus protease inhibitor cocktail and passed through a 27-gauge needle (Becton Dickinson, Franklin Lakes, NJ). The cell lysate was centrifuged at 14,500 x g for 10 min at 4°C in a microcentrifuge to obtain postnuclear supernatant. Proteins in the postnuclear supernatant were separated on 4-12% NuPage (Invitrogen) and transferred onto nitrocellulose membrane for immunoblotting. For detection of proteins on the membrane, horseradish peroxidase (HRP)-conjugated secondary antibodies and the Super Signal West Pico System (Thermo Fisher, Rockford, IL) were used. [00147] Microtubule polymerization assay

[00148] Ten T75 flasks at about 90% confluency were incubated in 10% FBS -containing medium for 20 hr. Cells were harvested with 0.05% Trypsin-EDTA, washed once in PBS followed by a wash with PMEE buffer. The cells were then resuspended in an equal volume of ice-cold homogenizing PMEE buffer [HG buffer: PMEE buffer plus 1 mM dithiothreitol, 1 mM 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, lx protease inhibitor cocktail and 0.5 mM ATP/MgS04] and passed through a 27-gauge needle. The cell lysate was centrifuged at 14,500 x g for 10 min at 4°C in a microcentrifuge. The supernatant was then ultracentrifuged at 192,000 x g for 30 min at 4°C to obtain a highspeed cell cytosol fraction (4.0 mg/ml protein). None, 50 μΜ tianeptine, or 20 μΜ taxol was added along with 1 mM GTP/MgS04 to 500 μΐ high-speed cell cytosol. The mixtures were incubated, with frequent inverting, at room temperature for 20 min under light for microtubule polymerization and were layered over 200 μΐ of 12.5%/25% sucrose cushions in a 700-μ1 ultracentrifuge tube and spun at 192,000 x g at 25°C for 45 min. An aliquot of the supernatant was saved and the rest of supernatant and sucrose cushion were removed in a stepwise manner by vacuum suction and cotton swab wiping, until only the microtubule pellet was left. The microtubule pellet was subsequently resuspended in 160 μΐ HG buffer. Both the supernatant and pellet were analyzed by Western blotting using anti-a-tubulin antibody.

[00149] Statistical Analysis

[00150] All quantifications in immunocytochemistry experiments were carried out at least in three separate experiments (n = 3) in each group unless stated otherwise. Data are presented as mean + SEM. Analyses of statistical significance were performed using student' s T test with 2 tails to establish statistical significance between experimental groups at the p<0.05 levels.

[00151] Results

[00152] Tianeptine causes formation of bundled microtubules and aberrant mitotic spindles in PC12 cells

[00153] The pheochromocytoma cell line PC12 was treated for 18 hr with control (H₂0), tianeptine (100 μΜ), or paclitaxel (50 μΜ) and microtubules were visualized by

immunocytochemistry using anti-a-tubulin antibody. -99% of control cells showed wellspread interphase microtubules (FIG. 1A) while less than 1 % of them showed mitotic spindles. On the other hand, most tianeptine-treated cells lost their normal interphase microtubule networks and, instead, showed bundled microtubules at the cell periphery (FIG. IB).

[00154] 88.22%+1.06 of tianeptine-treated cells showed bundled microtubules while none of control cells showed microtubule bundling (n=3: three independent experiments [100 cells per experiment], p<0.001, FIG. 1G).

[00155] In addition to microtubule bundling, the rest (-11%) of tianeptine-treated cells formed aberrant mitotic spindles, such as single (FIG. 1C) or multiple (FIG. ID) microtubule aster(s), sparse metaphase spindles (FIG. IE) and incomplete abscission (FIG. IF). [00156] Cells were treated twice with 100 μΜ tianeptine at a 12-hr interval to inhibit cell-cycle progression of the cells that escaped the initial impact of tianeptine because they were at cell-cycle stages insensitive to tianeptine. This double treatment caused not only more severe aberration of mitotic spindles but also massive microtubule bundling at the cell periphery (FIG. 1H).

[00157] Conversely, paclitaxel-treated cells (>95%) showed tubulin aggregates scattered in the cytoplasm (FIG. II).

[00158] These results show that tianeptine disturbs microtubule organization in PC 12 cells as paclitaxel does.

[00159] Bundled microtubules in tianeptine-treated cells are resistant to cold-induced depolymerization

[00160] Tianeptine causes formation of aberrant microtubules by affecting microtubule dynamics (assembly/disassembly). Incubation of live cells on ice causes microtubule

depolymerization in a time-dependent manner. Thus, cell incubation on ice is used to examine microtubule stability and dynamics.

[00161] Cells were treated for 18 hr with control (H₂0) or tianeptine (100 μΜ) prior to incubation on ice for 0, 10, 20 and 30 min. Then, the cells were fixed in -20°C methanol and immunostained using anti-a-tubulin antibody. Free cytosolic tubulins generated by microtubule depolymerization are usually not detected by immunocytochemistry because those are diffused out of cell that is permeabilized by methanol. Control cells showed tubulin fibrils throughout the cytoplasm prior to incubation on ice (FIG. 2A).

[00162] At 10 min after incubation on ice, there were few tubulin fibrils remaining in the cytoplasm of control cells while some were detected at the cell periphery (FIG. 2B).

[00163] In addition, the overall amounts of tubulins in the cytoplasm were decreased. The decrease is attributed to loss of free tubulins generated by microtubule depolymerization to outside of cells. At 20 min after incubation on ice, very few microtubules remained in the cytoplasm (FIG. 2C), showing that microtubules in control cells are completely depolymerized between 10 and 20 min on ice. On the other hand, bundled microtubules in tianeptine-treated cells were not depolymerized as quickly as those in control cells. At 0 min on ice, tianeptine-treated cells showed massive microtubule bundling at the cell periphery while few microtubules were present at the cell center (FIG. 2E).

[00164] Bundled microtubules at the cell periphery were not depolymerized after 10-min incubation on ice (FIG. 2F).

[00165] Even 20 min after incubation on ice, large amounts of bundled microtubules still remained at the cell periphery while some were depolymerized (FIG. 2G).

[00166] However, 30-min incubation on ice caused microtubule depolymerization in tianeptine-treated cells but not as much as in control cells (FIG. 2H).

[00167] The average intensities of microtubules at the cell periphery of cells incubated on ice were measured in the presence and absence of tianeptine. The average intensities of the peripheral microtubules in control cells after 10-min incubation on ice were dropped to 31.7+6.3% of those of the cells fixed without cold incubation (FIG. 21).

[00168] The average intensities of control cells remained around 40% throughout 30-min incubation on ice. In contrast, the average intensities of the peripheral microtubules in tianeptine - treated cells was not significantly decreased until 30 min on ice and then decreased to 73.0+3.64 of those of the cells fixed without cold incubation.

[00169] These results show that bundled microtubules in tianep tine-treated cells are very stable (less dynamic) and not easily depolymerized. Given that cell cycle progression entirely depends on microtubule disassembly and assembly, in other words, microtubule dynamics, the stable microtubule bundles formed by tianeptine treatment thus interfere with cell cycle progression in PC12 cells.

[00170] Tianeptine does not stabilize microtubules directly

[00171] The cold-resistant stability of bundled microtubules in tianeptine-treated cells was examined to determine whether tianeptine stabilizes polymerized microtubules directly as paclitaxel does. This was determined by in vitro microtubule polymerization assay. Endogenous tubulins in the cytosol from untreated cells were polymerized with 1 mM GTP/MgS0₄ plus none, 20 μΜ paclitaxel, or 50 μΜ tianeptine at 37°C for 20 min and spun through 12.5%/25% sucrose layers at -161,000 x g at 27°C for 45 min. The supernatant and pellet were processed for immunoblotting using anti-a-tubulin antibody. All of the paclitaxel-treated tubulins were polymerized and appeared in the pellet. On the other hand, addition of tianeptine did not increase the amount of polymerized microtubules in the pellet compared to no treatment (n=2, FIG. 2J), showing that tianeptine does not increase the stability of polymerized microtubules directly. Thus, tianeptine does not cause formation of microtubule bundling ex vivo by directly stabilizing microtubules.

[00172] CRMP2 coats aberrant mitotic spindles

[00173] Since tianeptine did not have a direct effect on microtubule stability, it was then determined whether it was possible that tianeptine affected the level and/or intracellular distribution of protein(s) involved in microtubule stabilization or microtubule organization.

[00174] The protein level and intracellular distribution of CRMP2, a microtubule stabilizer that is up-regulated in neurons treated with tianeptine, were examined. PC 12 cells treated with tianeptine (100 M, 18 hr) were processed to obtain cytosols to quantify the protein levels of CRMP2 by immunoblotting. The protein levels of CRMP2 were not different between untreated and tianeptine-treated PC 12 cells. The intracellular distribution of CRMP2 was, then, examined by immunocytochemistry using antibodies to a-tubulin and CRMP2 in untreated and tianeptine- treated cells. In untreated cells, CRMP2 did not show a significant colocalization with microtubules (FIG. 3A). [00175] The extent of colocalization of CRMP2 with microtubules, specifically aberrant mitotic spindles, significantly increased in tianeptine-treated cells (colocalization

coefficients.73+0.02) compared to untreated cells (colocalization coefficients.51+0.02) (n=30 cells, p<0.001, FIG. 3C). This shows that tianeptine treatment enhances association of the microtubule stabilizer, CRMP2, with microtubules, which appear to contribute to formation of aberrant mitotic spindles.

[00176] Tianeptine rapidly decreases the protein level of cytoplasmic dynein

[00177] Also examined were the protein levels and intracellular distribution of the microtubulebased motor, cytoplasmic dynein and the dynein activator, dynactin, both of which play important roles in microtubule organization during interphase and mitosis. Cytosols obtained from cells treated without (control) or with tianeptine (100-400 μΜ) for 18 hr were used for immunoblotting to detect cytoplasmic dynein (DIC: dynein intermediate chain) and dynactin (pl50: dynactin sidearm). The protein levels of cytoplasmic dynein decreased to -40% of control at 100 μΜ and to near zero at higher concentrations (n=2, FIGS 4A, 4B). The decrease in the levels of cytoplasmic dynein in tianeptine-treated cells was confirmed by immunocytochemistry using anti-DIC antibody. The average intensities of immunostained cytoplasmic dynein in tianeptine (100 μM)-treated cells (FIG. 4B) were decreased to -39% (28.1+1.4, FIG. 4E) of those in untreated cells (72.6+2.5, FIG. 4C, p<0.0001).

[00178] On the other hand, dynactin was decreased to -85% of control at 100 μΜ tianeptine and -40% at 300 μΜ tianeptine (n=2) (FIG. 4A). Also examined were the protein levels of a- tubulin and kinesin-1 (KHC: kinesin heavy chain), a- tubulin in cells treated with 100 μΜ tianeptine was decreased to -65% compared of control and to <10% at 300 μΜ. Kinesin-1 was also decreased to -50% of control at 100 μΜ or higher concentrations of tianeptine.

[00179] Interestingly, dynactin, a microtubule-associated protein that mediates stable binding of cytoplasmic dynein to microtubules showed high levels of colocalization (n=30 cells, colocalization coefficients.66+0.02) with both bundled microtubules and aberrant mitotic spindles (FIG. 5A).

[00180] The association of dynactin with bundled microtubules and aberrant mitotic spindles was increased in cells treated twice with 100 μΜ tianeptine at a 12-hr interval (n=30 cells, colocalization coefficient=0.82+0.01) (FIG. 5B).

[00181] These results show that reduction of cytoplasmic dynein in tianeptine-treated cells causes dispersion of dynactin along microtubules and mitotic spindles and that dispersion contributes to formation of microtubule bundles and aberrant mitotic spindles.

[00182] Dynactin disassembly that inhibits cytoplasmic dynein causes microtubule bundling [00183] The significant reduction of cytoplasmic dynein in tianeptine-treated cells inhibits cytoplasmic dynein/dynactin-dependent microtubule organization, such as anchoring of microtubules to the centrosomes. Over-expression of dynamitin that disassembles dynactin complex disrupts dynein/dynactin-dependent microtubule organizations during interphase and mitosis.

[00184] PC12 cells were transfected with GFP tag alone or GFP-tagged dynamitin (green) and stained with an ti- a- tubulin (red) antibody. Cells over-expressing GFP-dynamitin showed microtubule bundles (n=20 cells, FIG. 5D) while GFP-expressing cells did not (n=20 cells, FIG. 5C).

[00185] These results show that loss of cytoplasmic dynein in tianeptine-treated cells blocks cytoplasmic dynein/dynactin-mediated microtubule organization, resulting in formation of bundled microtubules and aberrant mitotic spindles.

[00186] Tianeptine-treated cells show abnormal nuclei

[00187] Also determined was whether tianeptine treatment affected the morphology of nucleus because the integrity of nucleus depends on microtubule organization.

[00188] PC12 cells were treated for 18 hr with none (FIG. 6A), 50 μΜ paclitaxel (FIG. 6B), or 100 μΜ tianeptine (FIG. 6C) and their chromosomes in the nucleus were visualized with propidium iodide. 48.0%+2.5 of the cells treated with 50 μΜ paclitaxel (18 hr) showed chromosome fragmentation and polyploid while none of control cells did (n=3 experiments, p<0.001, FIGS 6B, 6D).

[00189] Moreover, the number of paclitaxel-treated cells was increased by -2.0 fold compared to untreated cells (n=3, p<0.02, FIG. 6E), showing that paclitaxel treatment causes polyploidy and makes PC 12 cells more aggressive and proliferative.

[00190] In contrast, tianeptine treatment did not cause polyploidy but nuclear collapse and inclusions. The percent of live cells was decreased by tianeptine treatment to -63% of control (n=3, p<0.05, FIG. 6E).

[00191] Tianeptine-treated cells also showed abnormal nuclei with decondensed chromosomes (FIG. 6C), indicative of defective mitosis.

[00192] Also performed were BrdU staining to label dead cells. The percent of dead cells was increased by -11 folds in PC12 cells treated with tianeptine (-26.21+5.9%) compared to that in untreated cells (~2.27%+0.6) (n=3, p<0.02, FIG. 6F).

[00193] In contrast, few dead cells were found after treatment with paclitaxel (50 M, 18 hr).

[00194] These results show that tianeptine treatment interferes with cell cycle progression and causes nuclear collapse and eventually apoptosis.

[00195] Tianeptine increases aggregation of lamin-Bl

[00196] Since tianeptine treatment causes apoptosis in PC12 cells (FIG. 6), also examined was whether tianeptine caused aggregation of the nuclear membrane protein, lamin-B l, which indicates dying cells.

[00197] Cells treated with none (FIG. 7A) or 100 μΜ tianeptine (FIG. 7B) for 18 hr were permeabilized with 0.1% Triton X- 100 and immunostained using anti-lamin-Bl antibody. Compared to untreated cells (0.4%+0.0) (n=176, FIG. 7C), 75.6%+0.0 of tianeptine-treated cells showed lamin-B l aggregation (n=180, p<0.0001, FIG. 7C).

[00198] Thus, tianeptine treatment triggers apoptosis in PC12 cells.

[00199] Tianeptine causes apoptotic protein degradation while stabilizing p53 in PC 12 Cells [00200] Cytosols were extracted from cells treated with none (control), 50 μΜ paclitaxel, or 100 μΜ tianeptine for 18 hr and used for immunoblotting using antibodies against p53 (tumor suppressor), poly ADP-ribose polymerase (PARP), caspase 3 and lamin-B l. Compared to untreated cells, paclitaxel treatment decreased the protein level of p53 by -80% while it had no effect on PARP, caspase 3 and lamin-Bl (FIG. 7D).

[00201] In contrast, tianeptine treatment increased p53 slightly and decreased 116kD PARP significantly. However, tianeptine did not decrease the protein levels of caspase 3 and lamin-B l. These results show that tianeptine treatment causes specific degradation of PARP in addition to cytoplasmic dynein while not affecting the protein levels of caspase 3 and lamin-B 1. Moreover, tianeptine treatment stabilizes the tumor suppressor, p53. Thus, tianeptine makes PC12 cells more sensitive to p53-mediated apoptosis while paclitaxel should make the cells resistant to the apoptosis.

[00202] Tianeptine causes microtubule bundling and specific degradation of cytoplasmic dynein in human neuroblastoma cells

[00203] Also examined were the effects of tianeptine treatment on another neural crest-derived tumor cell line, SH-SY5Y (human neuroblastoma). The microtubule organization in tianeptine- treated SH-SY5Y cells were examined by immunocytochemistry using anti-a-tubulin antibody. 85.0%+0.7 of tianeptine-treated cells (n=200 cells, FIGS 8B, 8C) showed microtubule bundling and aberrant mitotic spindles while <2.0% of control did (n=200 cells, p<0.0001, FIGS 8A, 8C).

[00204] Cytosols obtained from SH-SY5Y cells treated with or without tianeptine (100 μΜ) for 18 hr were used for immunoblotting to detect cytoplasmic dynein (DIC), p53, dynactin (pl50), PARP, kinesin-1 (KHC),a-tubulin and lamin-B l (n=2, FIG. 8D).

[00205] The protein levels of cytoplasmic dynein (DIC) and dynactin (pl50) were significantly decreased by tianeptine treatment to <10% of control while those of p53 were slightly increased. Anti-PARP antibody did not recognize 116kD PARP in SH-5YSY cells but a 65kD protein band, instead. The 65kD PARP band was decreased by tianeptine treatment to <20% of that in control. The protein levels of kinesin-1 (KHC), a-tubulin and lamin-Bl were slightly decreased in tianeptine-treated cells. Thus, tianeptine causes microtubule bundling, degradation of cytoplasmic dynein/dynactin/PARP (65kD) while stabilizing p53 in SH-SY5Y cells as it does in PC12 cells.

[00206] Tianeptine does not cause apoptosis in rat embryonic cortical neurons

[00207] Also examined was whether tianeptine caused apoptosis in primary cell cultures.

[00208] Embryonic cortical neurons were treated with 100 μΜ tianeptine for 24 hr and examined whether neurons showed any apoptotic phenotype or neurite atrophy in response to tianeptine treatment. E18 rat embryonic cortical neurons were differentiated for 14 days in B27- supplemented neurobasal medium and treated with none (FIG. 9 A) or 100 μΜ tianeptine (FIG. 9B) in the neurobasal medium for 24 hr.

[00209] Then, the neurons were fixed with 3.7% paraformaldehyde and immunostained using anti-a-tubulin antibody and rhodaminephalloidin (F-actins). The morphology of neurons was analyzed with respect to the total length of neurites and the number of neurite branches per neuron. Dying neurons were supposed to show neurite atrophy (shortening and loss of neurites). Neurons treated with tianeptine, however, did not show neurite atrophy but formed 3.7-fold longer neurites and 3.0-fold more branches than control (n=20 neurons, P<0.0001, FIGS 9C, 9D).

[00210] These results show that tianeptine does not cause apoptosis in embryonic cortical neurons at the concentration that inhibits microtubule dynamics, thus causing apoptosis, in pheochromocytoma and neuroblastoma cells.

[00211] Tianeptine causes specific degradation of cytoplasmic dynein and increases the protein levels of tumor suppressors

[00212] Specific and rapid degradation of cytoplasmic dynein was observed in

pheochromocytoma PC- 12 cells treated with 1 μΜ tianeptine. Mock- treated cells showed a high level of cytoplasmic dynein while cells treated with 1, 5, 10 and 50 μΜ Tianeptine, the band intensity of dynein became very weak and at 100 μΜ there was none (FIG. 10).

[00213] The opposite patterns were observed for the tumor suppressor proteins, p53 and p27 (FIG. 10). In mock-treated cells, there were weak signals of p53 and p27. In the case of p53, the signals were progressively stronger as the concentration of tianeptine was increased. In contrast, the protein level of p53 was decreased to almost nil in cells treated with 20 μΜ paclitaxel (not shown).

[00214] As for p27, a weak signal in mock- treated cells was followed by signals of increasing intensity as the concentration of tianeptine was increased. The protein band intensity of tubulin was strong in mock-treated cells and those treated with 1 μΜ Tianeptine. 5 and 10 μΜ tianeptine caused a slight decrease in the levels of tubulins while there were further decreases in the tubulin levels in cells treated with 50 and 100 μΜ tianeptine. The very low protein levels of cytoplasmic dynein in cells treated with 1-100 μΜ tianeptine illustrates the ability of tianeptine to cause rapid degradation of cytoplasmic dynein in pheochromocytoma PC-12 cells. The rapid loss of cytoplasmic dynein should cause significant defects on cell cycle progression. Furthermore, the increase in the protein levels of p53 and p27 during the increase in the doses of tianeptine indicates that tianeptine can increase and/or stabilize p53 and p27, the tumor suppressor proteins that aid in preventing proliferation of pheochromocytoma tumor cells. Conversely, paclitaxel exerts the opposite action on p53, it caused degradation of p53. The reduction of p53 in paclitaxel-treated cells helps cells evade the cell-cycle arrest caused by microtubule disorganization. [00215] Discussion

[00216] Tianeptine inhibited microtubule dynamics required for cell-cycle progression during mitosis in the pheochromocytoma PC12 and neuroblastoma SH-SY5Y cells in a different way from paclitaxel.

[00217] Tianeptine is well tolerated and has few side effects on autonomic, cardiovascular, attention, sexual, learning and memory functions. Tianeptine increased microtubule bundling in both PC12 and SHSY5Y cells (FIG. 1 A - FIG. II, FIG. 8A - FIG.8D).

[00218] Microtubule bundling not only inhibits growth, angiogenesis and invasion of tumors cells but also causes apoptosis in some tumor cells. Microtubule bundling is caused by specific and rapid degradation of cytoplasmic dynein in tianeptine-treated PC 12 and SH-SY5Y cells (FIG. 4A - FIG. 4E, FIG. 8A - FIG. 8D).

[00219] The degradation of cytoplasmic dynein should cause accumulation of microtubules at the cell periphery because cytoplasmic dynein and its activator, dynactin, anchor microtubules to centrosomes, thus establishing a radial microtubule array during interphase. Over-expression of dynamitin that inhibits cytoplasmic dynein also induced microtubule bundling in PC 12 cells as tianeptine did. The reduction or inhibition of cytoplasmic dynein results in release of microtubules from the centrosomes and accumulation of unanchored microtubules at the cell periphery. The stability of bundled microtubules in tianeptine-treated cells was examined by exposing the cells to cold temperature. However, the unanchored microtubules accumulated at the cell periphery were not depolymerized by cold temperature. This showed that unanchored microtubules are not simply accumulated at the cell periphery and may be stabilized by a mechanism activated by tianeptine treatment.

[00220] Tianeptine treatment also increased association of collapsin response mediator protein- 2 (CRMP2), a microtubule stabilizer, with mitotic spindles. While not wishing to be bound by theory, it is believed that tianeptine treatment activates PI3K/Akt kinases and thus enhances the binding of CRMP2 to microtubules, which contributes to formation of aberrant mitotic spindles (extensively elongated mitotic spindles) that were observed in tianeptine-treated cells. In addition, the CRMP2-mediated microtubule elongation is now believed to contribute to neurite outgrowth in tianeptine-treated cortical neurons (FIG. 9).

[00221] Tianeptine-treated PC 12 cells also showed several apoptotic pheno types, such as protein degradation (except of the tumor suppressor, p53), nuclear rupture and inclusions and aggregation of lamin-B l, which was accompanied with an increase in the number of dying cells. Given that caspase 3 is not degraded by tianeptine treatment, caspase 3 may not be involved in the tianeptine-induced apoptosis. In contrast to tianeptine, paclitaxel did not cause apoptotic protein degradation while increased p53 degradation. Paclitaxel increased chromosome fragmentation (polyploidy) and even cell proliferation by ~2 fold. These results show that tianeptine can trigger apoptosis of PC 12 cells while paclitaxel makes pheochromocytoma cells more aggressive. Tianeptine causes the same types of mitotic defects and apoptosis in the neuroblastoma SH-SY5Y cells because tianeptine caused rapid degradation of cytoplasmic dynein and microtubule bundling in the cells. Since tianeptine did not cause apoptosis in primary cell cultures, its apoptotic action should not affect non-proliferating cells.

[00222] These results also show an anti-tumor activity of tianeptine against paclitaxel-resistant cancer cells, such as pheochromocytoma and neuroblastoma cells. Tianeptine causes microtubule bundling that prevents proliferation, migration and invasion of tumor cells. In addition, keeping p53 intact will make tumor cells more vulnerable to chemotherapy.

[00223] The anti-tumor properties of tianeptine provide a useful treatment of taxane-resistant cancers including malignant pheochromocytoma and neuroblastoma.

[00224] Further Examples

[00225] Therapeutic/Prophylactic Methods and Compositions

[00226] Also described herein are novel pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a therapeutic and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The carrier and composition can be sterile. The formulation will suit the mode of administration.

[00227] 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. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

[00228] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.

[00229] Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it is be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline is provided so that the ingredients are mixed prior to administration.

[00230] The amount of the therapeutic which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition and is determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the seriousness of the disease or disorder and is decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg kg body weight to 1 mg kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[00231] Combination Therapies

[00232] The compounds described herein may be used alone, in combination with one another, or as an adjunct to, or in conjunction with, other established antiproliferative therapies. Thus, the compounds may be used with traditional cancer therapies, such as ionization radiation in the form of .gamma.-rays and x-rays, delivered externally or internally by implantation of radioactive compounds and as a follow-up to surgical removal of tumors.

[00233] In another aspect, the compounds may be used with other chemotherapeutic agents useful for the disorder or condition being treated. These compounds may be administered simultaneously, sequentially, by the same route of administration, or by a different route.

[00234] Formulations and Administration

[00235] When used to treat or prevent such diseases, the active compounds and prodrugs may be administered singly, as mixtures of one or more active compounds, or in mixture or combination with other agents useful for treating such diseases and/or the symptoms associated with such diseases. The active compounds and prodrugs may also be administered in mixture or in combination with agents useful to treat other disorders or maladies, such as steroids, membrane stabilizers. The active compounds or prodrugs may be administered per se, or as pharmaceutical compositions comprising an active compound or prodrug.

[00236] Pharmaceutical compositions comprising the active compounds (or prodrugs thereof) may be manufactured by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically (see Remington's Pharmaceutical Sciences, 15.sup.th Ed., Hoover, J. E. ed., Mack Publishing Co. (2003)).

[00237] The active compound or prodrug may be formulated in the pharmaceutical compositions per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.

[00238] Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.

[00239] Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.

[00240] Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers and may contain added preservatives.

[00241] Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by any art-known technique, such as lyophilization and reconstituted prior to use.

[00242] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[00243] For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch,

polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate, lecithin). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings.

[00244] Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g. , lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, fractionated vegetable oils) and preservatives (e.g., methyl or propyl -p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound or prodrug, as is well known in the art. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the active compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides. For nasal administration or administration by inhalation or insufflation, the active compound(s) or prodrug(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[00245] The term "pharmaceutically acceptable salt" generally refers to any salt (e.g., obtained by reaction with an acid or a base) of a compound that is physiologically tolerated in the target animal (e.g., a mammal). Salts of the compounds may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p- sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia and the like. Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate,

benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,

cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2- hydroxyethanesulfonate, lactate, maleate, mesylate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate and the like. Other examples of salts include anions of the compounds compounded with a suitable cation such as Na+, NH₄+ and NW₄+ (wherein W is a C 1-4 alkyl group) and the like. For therapeutic use, salts of the compounds are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[00246] As used herein the terms "extended release dosage form," "controlled release dosage form," and "sustained release dosage form" and like expressions are used interchangeably and include dosage forms where the active drug substance or substances are released over an extended period of time. The term "extended" release should be understood in contrast to immediate release and, in particular, the term indicates that the formulation does not release the full dose of the active ingredient immediately after dosing. Such extended release dosage forms typically allow a reduction in dosing frequency as compared to that presented by a conventional dosage form such as a solution or an immediate release dosage form. The extended release forms may or may not comprise an immediate release component.

[00247] For prolonged delivery, the active compound(s) or prodrug(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection. The active ingredient may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the active compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the active compound(s).

[00248] Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver active compound(s) or prodrug(s).

[00249] The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[00250] Effective Dosages

[00251] The active compound(s) or prodrug(s), or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. The compound(s) may be administered

therapeutically to achieve therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also includes halting or slowing the progression of the disease, regardless of whether improvement is realized.

[00252] The amount of compound administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular active compound, etc. Determination of an effective dosage is well within the capabilities of those skilled in the art.

[00253] Effective dosages may be estimated initially from in vitro assays. For example, an initial dosage for use in animals may be formulated to achieve a circulating blood or serum concentration of active compound that is at or above an IC₅₀ of the particular compound as measured in an in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound is well within the capabilities of skilled artisans.

[00254] Initial dosages may also be estimated from in vivo data, such as animal models.

Animal models useful for testing the efficacy of compounds to treat or prevent the various diseases described above are well-known in the art. Dosage amounts will typically be in the range of from about 0.0001 or 0.001 or 0.01 mg/kg/day to about 100 mg kg/day, but may be higher or lower, depending upon, among other factors, the activity of the compound, its bioavailability, the mode of administration and various factors discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of active compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective local dosages without undue experimentation.

[00255] Preferably, the compound(s) will provide therapeutic or prophylactic benefit without causing substantial toxicity. Toxicity of the compound(s) may be determined using standard pharmaceutical procedures. The dose ratio between toxic and therapeutic (or prophylactic) LD₅₀/ED₅₀ effect is the therapeutic index (LD₅₀ is the dose lethal to 50% of the population and ED₅₀ is the dose therapeutically effective in 50% of the population). Compounds(s) that exhibit high therapeutic indices are preferred.

[00256] Kits

[00257] The compounds and/or prodrugs described herein may be assembled in the form of kits. In some embodiments, the kit provides the compound(s) and reagents to prepare a composition for administration. The composition may be in a dry or lyophilized form, or in a solution, particularly a sterile solution. When the composition is in a dry form, the reagent may comprise a pharmaceutically acceptable diluent for preparing a liquid formulation. The kit may contain a device for administration or for dispensing the compositions, including, but not limited to syringe, pipette, transdermal patch, or inhalant.

[00258] The kits may include other therapeutic compounds for use in conjunction with the compounds described herein. In some embodiments, the therapeutic agents are other anti-cancer and anti-neoplastic compounds. These compounds may be provided in a separate form, or mixed with the compounds described herein. The kits may include appropriate instructions for preparation and administration of the composition, side effects of the compositions and any other relevant information. The instructions may be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, or optical disc. [00259] The kits may also provide one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein. Optionally associated with such container(s) is a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[00260] All publications, including patents and non-patent literature, referred to in this specification are expressly incorporated by reference herein. Citation of the any of the documents recited herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00261] While the invention has been described with reference to various and preferred embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof.

[00262] Therefore, it is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.

Claims

CLAIMS What is claimed is:

1. A method for affecting one or more of: proliferation, migration and invasion of tumor cells, comprising contacting a tumor cell with an effective amount of a composition comprising tianeptine, sufficient to affect one or more of: proliferation, migration and invasion of tumor cells.

2. The method of claim 1, wherein the cells are taxane -resistant cancer cells.

3. The method of claim 1, wherein the tumor cells comprise malignant pheochromocytoma cells.

4. The method of claim 1, wherein the tumor cells comprise malignant neuroblastoma cells.

5. The method of claim 1, wherein the composition comprises tianeptine and at least one additional cancer therapeutic.

6. A method of claim 5, wherein the additional cancer therapeutic is selected from one or more of: chemotherapeutic drug, toxin, immunological response modifier, enzyme and radioisotope.

7. A method of claim 5, wherein the additional cancer therapeutic is selected from one or more of: paclitaxel, docetaxel, carboplatin, cyclophosphamide, doxorubicin and etoposide.

8. A method of claim 1, wherein the composition is capable of causing apoptosis of neuroblastoma cells.

9. A method of claim 1, wherein the composition is capable of affecting a cellular process selected from the group consisting of: increasing microtubule bundling, increasing anti- lamin-B 1 antibody levels, increasing apoptosis, inhibiting GTPase Ran, increasing protein degradation, increasing cytoplasmic dynein degradation, inhibiting collapsing response mediator protein-2 and/or increasing susceptibility to taxanes.

10. A method to affect at least one mammalian neuroblastoma cell, comprising contacting at least one mammalian neuroblastoma cell with at least an effective amount of a composition comprising tianeptine.

11. A method of claim 10, wherein the at least one mammalian neuroblastoma cell is derived from an animal selected from the group consisting of: mouse, rat, guinea pig, rabbit, cat, dog, monkey and human.

12. A method of claim 10, wherein the at least one mammalian neuroblastoma cell is cultured in vitro.

13. A method of claim 10, wherein the at least one mammalian neuroblastoma cell is an animal model.

14. A method of claim 10, wherein the at least one mammalian neuroblastoma cell is a human cell.

15. A method of ameliorating neuroblastoma in a mammal in need of such amelioration, comprising administering a composition comprising tianeptine.

16. A method of claim 15, wherein the neuroblastoma is present in a body location of the mammal selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

17. A method of claim 15, wherein the neuroblastoma is present in the brain.

18. A method of claim 10, wherein the composition comprises tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation.

19. A method of ameliorating neuroblastoma in a mammal in need of such amelioration, comprising administering a therapeutically-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to a mammal in need of neuroblastoma amelioration.

20. A method of claim 19, wherein the neuroblastoma is present in a body location selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

21. A method of claim 20, wherein the neuroblastoma is present in the brain.

22. A method of claim 20, which further comprises administering a cancer therapeutic selected from the group consisting of: chemotherapeutic drug, toxin, immunological response modifier, enzyme and radioisotope.

23. A composition of claim 20, which further comprises administering a cancer therapeutic selected from the group consisting of: paclitaxel, docetaxel, carboplatin, cyclophosphamide, doxorubicin and etoposide.

24. A method of claim 20, which further comprises physical removal of

neuroblastoma cells via a method selected from the group consisting of: surgery, aspiration, dissection, ablation and electromagnetic fluctuations.

25. A method of claim 20 or 22, wherein the mammal is selected from the group consisting of: mouse, rat, guinea pig, rabbit, cat, dog, monkey and human.

26. A method to cause neuroblastoma cell apoptosis, comprising contacting an apoptosis-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to at least one neuroblastoma cell.

27. A method of ameliorating taxane-resistant neuroblastoma in a mammal in need of such amelioration, comprising administering a therapeutically-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to a mammal in need of taxane-resistant neuroblastoma amelioration.

28. A method of claim 27, wherein the taxane-resistant neuroblastoma is present in a body location of the mammal selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

29. A method of claim 27, wherein the taxane-resistant neuroblastoma is present in the brain.

30. A method of claim 27, which further comprises administering a cancer therapeutic selected from the group consisting of: chemotherapeutic drug, toxin, immunological response modifier, enzyme and radioisotope.

31. A method of claim 27, which further comprises administering a cancer therapeutic selected from the group consisting of: paclitaxel, docetaxel, carboplatin, cyclophosphamide, doxorubicin and etoposide.

32. A method of claim 27, which further comprises physical removal of neuroblastoma cells via a method selected from the group consisting of: surgery, aspiration, dissection, ablation and electromagnetic fluctuations.

33. A method of inhibiting proliferation of a cell comprising contacting the cell with an amount of (lR,2R,3S,4S)-N4-(3-aminocarbonylbicyclo[2.2.1]hept-5-ene-2-yl)-5-fluoro- -N2- [3-methyl-4-(4-methylpiperazin-l-yl)]phenyl-2,4-pyrimidinediamine effective to inhibit its proliferation.

34. A method of inhibiting microtubule bundling in a cell in need thereof, comprising contacting the cell with an amount of tianeptine effective to inhibit microtubule bundling.

35. A method to affect at least one mammalian pheochromocytoma cell, comprising contacting at least one mammalian pheochromocytoma cell with at least an effective amount of tianeptine.

36. A method of claim 35, wherein the at least one mammalian pheochromocytoma cell is derived from an animal selected from the group consisting of: mouse, rat, guinea pig, rabbit, cat, dog, monkey and human.

37. A method of claim 35, wherein the at least one mammalian pheochromocytoma cell is cultured in vitro.

38. A method of claim 35, wherein the at least one mammalian pheochromocytoma cell is an animal model.

39. A method of claim 35, wherein the at least one mammalian pheochromocytoma cell is a human cell.

40. A method of ameliorating pheochromocytoma in a mammal in need of such amelioration, comprising administering an effective amount of a composition comprising tianeptine.

41. A method of claim 40, wherein the pheochromocytoma is present in a body location in the mammal selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

42. A method of claim 44, wherein the pheochromocytoma is present in the brain.

43. A method of claim 44, wherein the composition comprises tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof.

44. A method of ameliorating pheochromocytoma in a mammal in need of such amelioration, comprising administering a therapeutically-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to a mammal in need of pheochromocytoma amelioration.

45. A method of claim 52, wherein the pheochromocytoma is present in a body location in the mammal selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

46. A method of claim 53, which further comprises administering a cancer therapeutic selected from the group consisting of: chemotherapeutic drug, toxin, immunological response modifier, enzyme and radioisotope.

47. A method of claim 53, which further comprises administering a cancer therapeutic selected from the group consisting of: paclitaxel, docetaxel, carboplatin, cyclophosphamide, doxorubicin and etoposide.

48. A method of claim 53, which further comprises physical removal of

pheochromocytoma cells via a method selected from the group consisting of: surgery, aspiration, dissection, ablation and electromagnetic fluctuations.

49. A method of claim 53, wherein the mammal is selected from the group consisting of: mouse, rat, guinea pig, rabbit, cat, dog, monkey and human.

50. A method to cause pheochromocytoma cell apoptosis, comprising contacting an apoptosis-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to at least one pheochromocytoma cell.

51. A method of ameliorating taxane-resistant pheochromocytoma in a mammal in need of such amelioration, comprising administering a therapeutically-effective amount of tianeptine, or a pharmaceutically-acceptable analogue, metabolite, precursor, salt or formulation thereof to a mammal in need of taxane-resistant pheochromocytoma amelioration.

52. A method of claim 59, wherein the taxane-resistant pheochromocytoma is present in a body location in the mammal selected from the group consisting of: adrenal gland, brain, neck, chest, abdomen and back.

53. A method of claim 59, which further comprises administering a cancer therapeutic selected from the group consisting of: chemotherapeutic drug, toxin, immunological response modifier, enzyme and radioisotope.

54. A method of claim 59, which further comprises administering a cancer therapeutic selected from the group consisting of: paclitaxel, docetaxel, carboplatin, cyclophosphamide, doxorubicin and etoposide.

55. A method of claim 59, which further comprises physical removal of taxane- resistant pheochromocytoma cells via a method selected from the group consisting of: surgery, aspiration, dissection, ablation and electromagnetic fluctuations.

56. A method of any of claims 52-56, wherein the mammal is selected from the group consisting of: mouse, rat, guinea pig, rabbit, cat, dog, monkey and human.

57. A method of any one of the preceding claims, wherein the composition comprising tianeptine comprises predominantly the (R)-enantiomer of tianeptine and is substantially free of the (S)-enantiomer of tianeptine.

58. A method of any one of the preceding claims, wherein the composition comprising tianeptine comprises predominantly the (S)-enantiomer of tianeptine and is substantially free of the (R)-enantiomer of tianeptine.

59. A method of any one of the preceding claims, wherein the composition comprising tianeptine comprises a mixture of the (R)- and (S)-enantiomers of tianeptine.