USE OF NON-PEPTIDIC NK1 RECEPTOR ANTAGONISTS FOR THE PRODUCTION OF APOPTOSIS IN TUMOR CELLS

Abstract

Substance P antagonists and, in particular, non-peptidic NK1 receptor antagonists are useful for the treatment of cancer and, more specifically, human melanoma, neuroblastoma, glioma, human Hodgkin's lymphoma KM-H2, lymphoblastic leukemia, human rhabdomyosarcoma, human breast carcinoma, human Burkitt's lymphoma, human lung carcinoma, human Ewing's sarcoma, human glioma and human osteosarcoma.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a Division of U.S. Ser. No. 11/721,256, filed Sep. 16, 2008, which is a National Phase conversion under 35 U.S.C. §371 of PCT/ES2005/000068, filed Feb. 10, 2005, which claims benefit of priority from Spanish Application No. ES200400424, filed Feb. 11, 2004, the disclosure of which are each incorporated herein by reference. PCT International Application was published in the Spanish language.

FIELD OF THE INVENTION

[0002] The invention involves the use of P substance antagonists, specifically non-peptide NK1 receptor antagonists, in the treatment of human cancers, explicitly on melanoma, neuroblastoma, glioma, Hodgkin's-KM-H2 lymphoma, lymphoblastic leukemia, Rhabdomyosarcoma, breast cancer, Burkitt's lymphoma, lung cancer, Ewing's sarcoma and human osteosarcoma.

BACKGROUND OF THE INVENTION

[0003] Substance P is a natural undecapeptide from the Tachykinins family and is used for its rapid stimulant action on smooth muscle tissue. More specifically, Substance P is an active pharmacological neuropeptide that is produced in mammals. It was originally isolated in the intestine and possesses an amino acid sequence that has been described by D. F. Veber, in the patent U.S. Pat. No. 4,680,283. The implication of Substance P, like in other Tachykinins, is seen in the physiopathology in a large number of illnesses that are well demonstrated in the bibliography.

[0004] Substance P receptor is a member of the super family of G-protein-coupled receptors. The neuropeptide receptor of Substance P (NK-1) is well distributed in the nervous system of mammals (especially in the cerebrum and spinal cord) the circulatory system and in the peripheral tissues (especially in the duodenum and in the jejunum) and is involved in the regulation of diverse biological processes.

[0005] The central and peripheral action of the Tachykinins in mammals have been associated with various inflammatory conditions such as migraines, rheumatoid arthritis, asthma, and intestinal inflammatory disease, as well as in the mediation of nauseous reflexes, and the regulation of CNS central nervous system disorders such as Parkinson's disease. (Neurosci. Res., 1996, 7, 187-214), anxiety (Can. J. Phys.; 1997, 612-621) and depression (Science, 1998, 281, 1640-1645).

[0006] In the article titled "Tachykinin Receptor and Tachykinin Receptor Antagonists", by J. Auton, in Pharmacol.; 1993, 13, 23-93, the use of antagonists of Substance P have been evidenced in the treatment of headaches, especially migraines, Alzheimer's disease, multiple sclerosis, attenuation of the syndrome in the absence of opiates, cardio vascular changes, edemas, such as those provoked by burns, in chronic inflammatory illnesses like rheumatoid arthritis, asthma, hyperactive bronchials, and other respiratory illnesses including allergic rhinitis, etc.

[0007] Also, U.S. Pat. No. 5,972,938 describes a method for the treatment of a psychoimmunological disorder or psychomotor by way of the administration of an NK1 receptor antagonist.

[0008] The article published in Nature, 2000, 405 (6783), 180-183 details the activity in rats lacking NK-1 receptors and shows a decrease in the beneficial effects of morphine. Consequently, the NK-1 antagonist receptors can be used in the treatment of breaking certain drug addition habits such as those associated with opiates, nicotine as well as in the reduction of abuse and abstinence from the drugs.

[0009] The article in Life Sci.; 2000, 67(9), 985-1001 describes the Astrocytes express functional receptors for various neurotransmitters in the reception of Substance P. The cerebral tumors of malignant glials derived from Astrocytes unchain under the action of the Tachykinins mediating the NK-1 receptors in the secretion of soluble mediators that augment the speed of reproliferation. Consequently, the selective antagonists of NK-1 can be very useful therapeutic agents in the treatment of malignant gliomas and for the treatment of cancer.

[0010] Additionally, the New Journal of Medicine, 1999, 340, 190-195, states that the use of a selective NK1 receptor induces the reduction of vomiting by employing cisplatin.

[0011] In the article published in the International Journal of Cancer by Antal Orosz et al. 1995, 60, 82-87, the use of diverse peptide antagonists in Substance P (SP) is described in the inhibition of the proliferation of lung cancer cells. (Ex. in designated cells NCI-H69). Equally as important is the article published in Cancer Research, 1994, 54, 3602-3610, describing another antagonist of Substance P (SP) as well as other peptides capable of the inhibition of the growth of various in-vitro lines in cancerous lung cells (ex. Designated cells NCI-H510, NCI-H345, and SHP-77).

[0012] The patent EP 773026 (Pfizer) states the use of non-peptide NK1 receptor antagonists in the treatment of breast cancer, particularly in the treatment of small lung cancers in APUdoma, neuro endocrinic tumors, and small extra lung carcinomas.

[0013] Additionally in the WO 2001001922 patent the use of NK1 receptors in the treatment of adenocarcinoma is described, most specifically in prostatic carcinomas. Giardina, G.; Gagliardi S. and Martinelli M. review the most recent patents about the NK1, NK2 and NK3 receptors in "Antagonists at the neurokinin receptors-Recent patent literature" (IDrugs 2003; 6(8): 758-772). The authors describe the action of the molecules of the most important world producers with a specific indication of the most noteworthy possible applications being used in the treatment of: depression, inflammation, anxiety, vomiting, Ulcerative colitis and other illnesses.

SUMMARY OF THE INVENTION

[0014] The objective of the current invention is the use of non-peptide NK1 receptor antagonists and Substance P for the production of apoptosis in breast cancer tumors. The tumor cells that the antagonists act on present a number of NK1 receptors that is superior to those in non tumor cells, composed of between 400% and 500% of the normal number of non tumor cells.

[0015] The tumor cells that the antagonists act on are selected from the group consisting of:

[0016] invasive primary and invasive malignant melanomas;

[0017] metastatic melanoma cells;

[0018] cells localized in ganglion lymph nodes--glioma cells--human breast cancer cells;

[0019] Acute lymphoblastic leukemia B cells;

[0020] Acute lymphoblastic leukemia T cells;

[0021] primary neuroblastoma cells--astrocytoma cells;

[0022] Burkitt's lymphoma cells;

[0023] Hodgkin's lymphoma cells;

[0024] Rhabdomyosarcoma cells;

[0025] small lung cancer cells;

[0026] Ewing's sarcoma cells; and

[0027] osteosarcoma cells.

[0028] They indicated the continuation in specific cells acted upon by the non-peptide NK1 receptor antagonists and substance P.

[0029] The tumor cells related to human melanoma on which the current antagonists act in cell lines are COLO 858 [ICLC, Interlab Cell Line Collection--CBA--Genova), MEL HO [DSMZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen] and COLO 679 [DSMZ].

[0030] The tumor cells related to the human glioma and the human neuroblastoma to which the antagonists act on in cell lines are the GAMG [DSMZ] and SKN-BE (2) [ICLC].

[0031] The tumor cells related to lymphoblastic leukemia which the current antagonists act on are human lymphoblastic leukemia cells B SD1 [DSMZ] and human lymphoblastic leukemia cells TBE-13 [DSMZ]. The tumor cells related to Burkitt's lymphoma on which the antagonists act in cell lines are CA-46 [DSMZ]. The tumor cells related to Hodgkin's lymphoma on which the antagonists act are KM-H2 [DSMZ]. The tumor cells related to human rhabdomyosarcoma on which the antagonists act in a cell line form are A-204 [DSMZ]. The tumor cells related to small human lung cancer cells on which the antagonists act in a cell line are COLO-677 [DSMZ]. The tumor cells related to human breast cancer on which the antagonists act in a cell line are MT-3 [DSMZ].

[0032] The tumor cells related to Ewing's sarcoma on which the current antagonists act in a cell line are MHH-ES-1 [DSMZ].

[0033] The tumor cells related to human osteosarcoma on which the current antagonists act in a cell line are MG-63 [ICLC].

[0034] One of the antagonists used is (2S,3S) 3-{[3,5-Bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine, commercially known as L-733060 (Sigma-Aldrich) and used in concentrations composed of between 5 μM and 50 μM.

[0035] Other compounds of antagonist non-peptide receptors NK1 and Substance P that can be used include:

[0036] vofopitant6GR-205171 (Pfizer);

[0037] eziopitant 6 CJ-11974 (Pfizer);

[0038] CP-122721 (Pfizer);

[0039] Aprepitant 6 MK 869 6 L-754030 (MSD);

[0040] L-758298 (MSD);

[0041] TAK-637 (Takeda/Abbot);

[0042] GW597599 (GSK);

[0043] GW679769 (GSK); and

[0044] R673 (Roche).

[0045] Lastly, the other objective on the current invention is the use of the non-peptide NK1 receptor and Substance P antagonists, such as the aforementioned indicators in the creation of a pharmaceutical composition for the treatment of cancer.

[0046] It is therefore an object to provide for the use of non-peptide NK1 receptor and Substance P antagonists to induce apoptosis in tumor cells of mammals.

[0047] It is also an object to provide for the use of non-peptide NK1 receptor and Substance P antagonists to induce apoptosis in tumor cells of mammals characterized in that the tumor cells that the antagonists act on present between 400% and 500% of the number of NK1 receptors as compared to those present in non-tumor cells.

[0048] It is a further object to provide for the use of non-peptide NK1 receptor and Substance P antagonists to induce apoptosis in tumor cells of mammals, characterized in that the tumor cells that the antagonists act on are selected among: invasive primary and invasive malignant melanomas; metastatic melanoma cells; cells localized in ganglion lymph nodes; glioma cells--human breast cancer cells; Acute lymphoblastic leukemia B cells; Acute lymphoblastic leukemia T cells; primary neuroblastoma cells; astrocytoma cells; Burkitt's lymphoma cells; Hodgkin's lymphoma cells; rhabdomyosarcoma cells; small lung cancer cells; Ewing's sarcoma cells; and osteosarcoma cells.

[0049] The antagonists act, for example, on tumor cells related to any of:

[0050] human melanoma cell lines COLO 858 [ICLC, Interlab Cell Line Collection--CBA--Genova), MEL HO [DSMZ, Deutsche Sammlung von Mikroorganismen undZelikulturen] and COLO 679 [DSMZ];

[0051] human glioma and neuroblastoma cell lines GAMG [DSMZ] and SKN-BE (2) [ICLC];

[0052] lymphoblastic leukemia cell lines B SD1 [DSMZ] and TBE-13 [DSMZ];

[0053] Burkitt's lymphoma cell line CA-46 [DSMZ];

[0054] Hodgkin's lymphoma cell line KM-H2 [DSMZ];

[0055] rhabdomyosarcoma cell line A-204 [DSMZ];

[0056] small cell lung cancer cell line COLO-677 [DSMZ];

[0057] breast cancer cell line MT-3 [DSMZ];

[0058] Ewing's sarcoma cell line MHH-ES-1 [DSMZ]; and

[0059] osteosarcoma cell line MG-63[ICLC].

[0060] It is a further object to induce apoptosis in tumor cells of mammals using of the antagonist (2S,3S) 3-{[3,5-Bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine, commercially known as L-733060 (Sigma-Aldrich). The antagonist L-733060 may be used in concentrations of between 5 μM and 50 μM.

[0061] It is a still further object to induce apoptosis in tumor cells of mammals by uses of a antagonist that is selected among some of the following compounds: vofopitant 6 GR-205171 (Pfizer); eziopitant 6 CJ-11974 (Pfizer); CP-122721 (Pfizer); Aprepitant 6 MK 869 6 L-754030 (MSD); L-758298 (MSD); TAK-637 (Takeda/Abbot); GW597599 (GSK); GW679769 (GSK); and R673 (Roche).

[0062] It is also an object to use non-peptide NK1 receptor and Substance P antagonists for the development of a pharmaceutical compound for the treatment of cancer.

BRIEF DESCRIPTIONS OF THE FIGURES

[0063] FIGS. 1A and 1B: variation in the time of the concentration of the cells SKN-BE (2) to growing concentrations of L-733,060 (1A) in the cellular growth inhibition of SKN-BE (2) (1B).

[0064] FIGS. 2A and 2B: variation in the time of the concentration of the cells COLO 858 to growing concentrations of L-733,060 (2A) in the cellular growth inhibition of COLO 858 (2B).

[0065] FIGS. 3A and 3B: variation in the time of the concentration of the cells MEL HO to growing concentrations of L-733,060 (3A) in the cellular growth inhibition of MEL HO (3B).

[0066] FIGS. 4A and 4B: variation in the time of the concentration of the cells COLO 679 to growing concentrations of L-733,060 (4A) in the cellular growth inhibition of COLO 679 (4B).

[0067] FIG. 5: variation in the time of the concentration of the cells SD1 to growing concentrations of L-733,060 (5A) in the cellular growth inhibition of SD1 (5B).

[0068] FIGS. 6A and 6B: variation in the time of the concentration of the cells KM-H2 to growing concentrations of L-733,060 (6A) in the cellular growth inhibition of KM-H2 (6B).

[0069] FIGS. 7A and 7B: variation in the time of the concentration of the cells MT-3 to growing concentrations of L-733,060 (7A) in the cellular growth inhibition of MT-3 (7B).

[0070] FIGS. 8A and 8B: variation in the time of the concentration of the cells MHH-ES-1 to growing concentrations of L-733,060 (8A) in the cellular growth inhibition of MHH-ES-1 (8B).

[0071] FIG. 9: variation in the time of the concentration of the cells MG-63 to growing concentrations of L-733,060 (9A) in the cellular growth inhibition of MG-63 (9B).

[0072] FIGS. 10A and 10B: variation in the time of the concentration of the cells GAMG to growing concentrations of L-733,060 (10A) in the cellular growth inhibition of GAMG (10B).

DETAILED DESCRIPTION OF THE INVENTION

[0073] In addition, a detailed explanation of how the activity was carried out was based on the current invention of each tested in cell lines. The following examples are provided only in order to illustrate the invention and thus they should not be construed as limiting.

Example 1

Cell Lines Related to Neuroblastoma

[0074] A Cell line of human neuroblastoma SKN-BE (2)(ICLC Interlab Cell Line Collection--CBA--Genova) was used.

[0075] This line was maintained in a culture of RPMI 1640 (GIBCO-BRL) supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0076] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half was refreshed every two days and the cells were treated with trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at a temperature of 37 C in humidified (95% air/5% CO₂).

[0077] Treatment with the NK1 receptor antagonists: The solutions of antagonist NK1 receptors (2S,3S)3-([3,5Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060) (Sigma-Aldrich, U.K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 20 μM) were studied with the objective of determining the IC₅₀.

[0078] The proliferation of cells was tested using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium]method, following the instructions established by (Cet) Titer 96 Aqueous One Solution Cell Proliferation Assay, Promega, (USA).

[0079] Methods of cellular proliferation: During the experiment, the cultivated cells were broken apart every 4-5 days by way of trypsinization and to test the cell viability the trypan blue method was used. The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0080] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0081] In T₀, 20 μl of MTS reagent was immediately added to the wells and the wells were read 90 minutes later. T₁ and T₂ were treated with different concentrations of (2.5 μM to 20 μM) of L-733,060 and were incubated during a period of 30 hrs. (first cellular duplication) (T₁) and 72 hrs. (second cellular duplication) (T₂).

[0082] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by testing the optical density, being directly proportional to the number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on an adequate curve based on the parameters.

[0083] Statistical Analysis: The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0084] Results: The results shown in FIG. 1A represent the variation in time of the concentration of cells SKN-BE (2) at growing concentrations of L-733,060.

[0085] FIG. 1B shows the percentage of inhibition of cellular growth of SKNBE (2) (at 30 hrs. and 72 hrs.) after the addition of growing concentrations of L-733,060 (2.5, 5, 10, 20 μM), at the first and second duplication times of the incubation. The non-continuous lines represent IC₅₀ at 30 and 72 hrs. The points on the graph represent the average value/typical deviation.

Example 2

Cell Lines Related to Melanomas

[0086] Cell lines related to melanomas COLO 858 (ICLC Interlab Cell LineCollection--CBA--Genova), MEL HO and COLO 679 (DSMZDeutsche Sammlung von Mikroorganismen and Zellkulturen) were used.

[0087] These cell lines were maintained in a culture of RPMI 1640 (GIBCOBRL) supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC, the ICLC and the DSMZ.

[0088] The cell lines were cultivated in 75 ml flasks (Falcon, Germany).

[0089] Half was renewed every two days and the cells were treated with trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at 37 C in humidified (95% air/5% CO₂).

[0090] The NK1 receptor antagonist, (2S,3S)3-([3,5Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine (L-733,060)(Sigma-Aldrich, U. K.) was dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. With the objective of determining the IC₅₀, different concentrations (2.5 μM to 50 μM) were studied.

[0091] The cellular proliferation was evaluated using the MTS method 1344,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], according to the instructions of use established by (Cet) Titer 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0092] Cellular Proliferation Method: During the experiment, the cultivated cells were broken apart every 4-5 days by way of trypsinization and to test the cell viability the trypan blue method was used. The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0093] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0094] 20 μl of MTS reagent was immediately added to the T₀ wells and they were read 90 minutes after. T₁ and T₂ were treated with different concentrations (2.5 μM to 50 μM) of L-733,060 and were incubated during a varying period in cell lines.

[0095] Line COLO 858: 48 h. (first cellular duplication) (T₁) and 96 hrs. (second cellular duplication) (T₂).

[0096] Line MEL HO: 24 hrs. (cellular duplication) (T₁) and 48 hrs. (second cellular duplication) (T₂).

[0097] Line COLO 679: 30 hrs. (cellular duplication) (T₁) and 72 hrs. (second cellular duplication) (T₂).

[0098] To study the cellular proliferation, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the plate samples with the (TECAN Spectra Classic) 492 nm. The quantity of MTS reagent was determined by measurement of the optical density, being directly proportional to the number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentrations to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on a curve suited to the parameters.

[0099] Statistical Analysis: The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0100] Results: The results are shown in FIGS. 2A, 2B (COLO 858), FIGS. 3A and 3B (MEL HO) and FIGS. 4A and 4B (COLO 679).

[0101] FIG. 2A represents the variation in the time of the concentration of cells COLO 858 to growing concentrations of L-733,060 (from 2.5 to 20 μM).

[0102] FIG. 3A represents the variation in the time of the concentration of cells MEL HO to growing concentrations of L-733,060 (from 10 to 30 μM).

[0103] FIG. 4A represents the variation in the time of the concentration of cells COLO 679 to growing concentrations of L-733,060 (from 20 to 50 μM).

[0104] In FIG. 2B, the inhibition of cellular growth is shown from COLO 858 (at 48 and 96 hrs.) after the addition of growing concentrations of L-733,060 (2.5, 5, 10, 20 μM). The discontinuous lines represent the IC₅₀ for 48 and 96 hrs. The points on the graph represent the value of the average value/typical deviation.

[0105] In FIG. 3B the inhibition of cellular growth is shown from MEL HO (at 24 and 48 hrs.) after the addition of growing concentrations of L-733,060 (10, 20, 25, and 30 μM). The discontinuous lines represent the IC₅₀ for 24 and 48 hrs. The points on the graph represent the average value/typical deviation.

[0106] In FIG. 4B the inhibition of cellular growth is shown from COLO 679 (at 30 and 72 hrs. after the addition of growing concentrations of L-733,060 (20, 30, 40, and 50 μM). The discontinuous lines represent the IC₅₀ for 30 and 72 hrs. The points on the graph represent the average value/typical deviation.

Example 3

Cell Lines Related to Lymphoblastic Leukemia

[0107] Cell lines related to human lymphoblastic leukemia were used, BSD1 (DSMZ) and T BE-13 (DSMZ).

[0108] These cell lines were maintained in a culture of 1640 supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0109] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO₂).

[0110] Treatment with antagonist NK1 receptors: The solutions of the antagonist NK1 receptors (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060) (Sigma-Aldrich, U.K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 25 μM) were studied in order to determine the IC₅₀.

[0111] The cellular proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0112] Method of Cellular Proliferation: The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0113] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0114] In T₀, 20 μl of MTS reagent was immediately added to the wells and they were read 90 minutes later. T₁ and T₂ were treated with different concentrations (2.5 μM to 25 μM) of L-733,060 and were incubated during a period of 30 hrs. (cellular duplication) (T₁) and 72 hrs. (second cellular duplication) (T₂).

[0115] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 180 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by optical density, being directly proportional in number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated with a curve suited to the parameters.

[0116] Statistical Analysis: The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0117] Results: The results shown in FIG. 5 represents the variation in time of the concentration of cells BSD1 to growing concentrations of L-733,060.

[0118] In FIG. 5, the inhibition of cell growth BSD1 is represented (at 30 and 72 hrs.) after the addition of growing concentrations of L-733,060 (2.5, 5, 10, 25 μM). The percentage of the inhibition for the first and second time of the duplication of the incubation is shown. The discontinuous lines represent the IC₅₀ at 30 and 72 hrs. The points on the graph represent the average value/typical deviation.

Example 4

Cell Line Related to Burkitt's Human Lymphoma

[0119] Linear cells related to human Burkitt's lymphoma were used with CA-46 (DSMZ).

[0120] This cell line was maintained in a culture of RPMI 1640 and supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0121] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days and the cells were treated with trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO₂).

[0122] Treatment with NK1 receptor antagonists: The solutions of the antagonist NK1 receptors (2S,3S)3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060) (Sigma-Aldrich, U. K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 25 μM) were studied to determine the IC₅₀.

[0123] The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0124] Method of Cellular Proliferation: The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates called T₀, T₁ and T₂.

[0125] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0126] In T₀, 20 μl of MTS reagent was immediately added to the wells and they were read 90 minutes later. T₁ and T₂ were treated with different concentrations (2.5 μM to 25 μM) of L-733,060 and were incubated during a period of 35 hrs. (cellular duplication) (T₁) and 72 hrs. (second cellular duplication) (T₂).

[0127] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by optical density, being directly proportional in number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (ICso) with L-733,060 was calculated on a curve suited to the parameters.

[0128] Statistical Analysis: The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0129] Results: At the highest concentrations, inhibition in cellular growth was produced and at the maximum dose, apoptosis.

Example 5

Cell Lines Related to Human Hodgkin's Lymphoma

[0130] A cell line related to human Hodgkin's lymphoma.KM-H2 (DSMZ) was used.

[0131] This cell line was maintained in a culture of RPMI 1640 and supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0132] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO₂).

[0133] Treatment with NK1 receptor antagonists: The solutions of the NK1 receptor antagonists (2S,3S)3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060)(Sigma-Aldrich, U. K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 20 μM) were studied to determine the IC₅₀.

[0134] The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0135] Method of Cellular Proliferation: The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0136] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both, T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0137] In T₀, 20 μl of MTS reagent was immediately added to the wells and they were read 90 minutes later. T₁ and T₂ were treated with different concentrations of (2.5 μM to 20 μM) of L-733,060 and were incubated during a period of 48 hrs. (cellular duplication) (T₁) and 96 hrs. (second cellular duplication) (T₂).

[0138] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 180 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by optical density, being directly proportional in number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on a curve suited to the parameters.

[0139] The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0140] Results: The results shown in FIG. 6A represent the variation in the time of the concentration of the cells KM-H2 with growing concentrations of L-733,060.

[0141] In FIG. 6B the inhibition of cell growth KM-H2 is represented (at 48 and 96 hrs.) after the addition of growing concentrations of L-733,060 (2.5, 5, 10, 20 μM). The percentage of the inhibition for the first and second time of the duplication of the incubation. The discontinuous lines represent the IC₅₀ at 48 and 96 hrs. The points on the graph represent the average value/typical deviation.

Example 6

Cell Lines Related to Human Rhabdomyosarcoma

[0142] A cell line related to human rhabdomyosarcoma A-204 (DSMZ) were used.

[0143] This cell line was maintained in a culture of Mc--Co-- supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0144] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days and the cells were treated with Trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO₂).

[0145] Treatment with antagonist NK1 receptors: The solutions of the antagonist NK1 receptors (2S,3S)3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060)(Sigma-Aldrich, U. K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 25 μM) were studied to determine the IC₅₀.

[0146] The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0147] Method of Cellular Proliferation: During the experiment, the cultivated cells were broken apart every 4-5 days by way of trypsinization and to test the cell viability the trypan blue method was used. The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0148] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0149] In T₀, 20 μl of MTS reagent was immediately added to the wells and were read 90 minutes later. T₁ and T₂ were treated with different concentrations (2.5 μM to 20 μM) of L-733,060 and were incubated during a period of 36 hrs. (first cellular duplication) (T₁) and 72 hrs. (second cellular duplication) (T₂).

[0150] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by testing the optical density, being directly proportional to the number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on an adequate curve based on the parameters.

[0151] Statistical Analysis: The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0152] Results: Cellular growth is inhibited at the highest concentrations and at the maximum dose, apoptosis.

Example 7

Cell Lines Related to Small Cell Lung Cancer

[0153] A cell line related to small cell lung cancer COLO-677 (DSMZ) was used. This cell line was maintained in a culture of RPMI 1640 supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0154] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days and the cells were treated with Trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO2).

[0155] Treatment with antagonist NK1 receptors: The solutions of the antagonist NK1 receptors (2S,3S)3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060)(Sigma-Aldrich, U. K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 25 μM) were studied to determine the IC₅₀.

[0156] The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0157] Method of Cellular Proliferation: During the experiment, the cultivated cells were broken apart every 4-5 days by way of trypsinization and to test the cell viability the trypan blue method was used. The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0158] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0159] In T₀, 20 μl of MTS reagent was immediately added to the wells and they were read 90 minutes later. T₁ and T₂ were treated with different concentrations (5 μM to 20 μM) of L-733,060 and were incubated during a period of 40 hrs. (first cellular duplication) (T₁) and 96 hrs. (second cellular duplication) (T₂).

[0160] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by testing the optical density, being directly proportional to the number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on an adequate curve based on the parameters.

[0161] Statistical Analysis The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0162] Results: Cellular growth is inhibited at the highest concentrations and at the maximum dose, apoptosis.

Example 8

Cell Lines Related to Human Breast Cancer

[0163] A cell line related to human breast cancer MT-3 (DSMZ) was used. This cell line was maintained in a culture of RPMI 1640 supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0164] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days and the cells were treated with Trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO2).

[0165] Treatment with NK1 receptor antagonists: The solutions of the NK1 receptor antagonists (2S,3S)3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060) (Sigma-Aldrich, U. K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 20 μM) were studied to determine the IC₅₀.

[0166] The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0167] Method of Cellular Proliferation: During the experiment, the cultivated cells were broken apart every 4-5 days by way of trypsinization and to test the cell viability the trypan blue method was used. The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0168] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0169] In T₀, 20 μl of MTS reagent was immediately added to the wells and they were read 90 minutes later. T₁ and T₂ were treated with different concentrations (2.5 μM to 20 μM) of L-733,060 and were incubated during a period of 30 hrs. (first cellular duplication) (T₁) and 72 hrs. (second cellular duplication) (T₂).

[0170] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by optical density, being directly proportional in number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on a curve suited to the parameters.

[0171] Statistical Analysis: The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0172] Results: The results shown in FIG. 7A represent the variation in the time of the concentration of cells MT-3 at growing concentrations of L-733,060.

[0173] In FIG. 7B, the inhibition of cell growth MT-3 is represented (at 30 and 72 hrs.) after the addition of increasing concentrations of L-733,060 (2.5, 5, 10, 20 μM). The percentage of the inhibition for the first and second time in the duplication of the incubation. The discontinuous lines represent the IC₅₀ at 30 and 72 hrs. The points on the graph represent the average value/typical deviation.

Example 9

Cell Lines Related to Ewing's Human Sarcoma

[0174] A cell lines related to Ewing's human sarcoma MHH-ES-1 (DSMZ) was used. This cell line was maintained in a culture of RPMI 1640 supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0175] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days and the cells were treated with Trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO2).

[0176] Treatment with NK1 receptor antagonists The solutions of the NK1 receptor antagonists (2S,3S)3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060) (Sigma-Aldrich, U.K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 25 μM) were studied to determine the IC₅₀.

[0177] The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0178] Method of Cellular Proliferation: During the experiment, the cultivated cells were broken apart every 4-5 days by way of trypsinization and to test the cell viability the trypan blue method was used. The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0179] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0180] In T₀, 20 μl of MTS reagent was immediately added to the wells and they were read 90 minutes later. T₁ and T₂ were treated with different concentrations (5 μM to 20 μM) of L-733,060 and were incubated during a period of 30 hrs. (first cellular duplication) (T₁) and 72 hrs. (second cellular duplication) (T₂).

[0181] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by optical density, being directly proportional in number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on a curve suited to the parameters.

[0182] Statistical Analysis: The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0183] Results: The results shown in FIG. 8A represent the variation in the time of the concentration of cells MHH-ES-1 at growing concentrations of L-733,060.

[0184] In FIG. 8B the inhibition of cell growth MHH-ES-1 is represented (at 30 and 72 hrs.) after the addition of growing concentrations of L-733,060 (5, 10, 15, 20, μM). The percentage of the inhibition for the first and second time of the duplication of the incubation. The discontinuous lines represent the IC₅₀ at 30 and 72 hrs. The points on the graph represent the average value/typical deviation.

Example 10

Cell Line Related to Human Osteosarcoma

[0185] A cell line related to human osteosarcoma MG-63 (ICLC) was used.

[0186] This cell line was maintained in a culture of MEN supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0187] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days and the cells were treated with Trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO2).

[0188] Treatment with NK1 receptor antagonists: The solutions of the NK1 receptor antagonists (2S,3S)3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060) (Sigma-Aldrich, U.K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 25 μM) were studied to determine the IC₅₀.

[0189] The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0190] Method of Cellular Proliferation: During the experiment, the cultivated cells were broken apart every 4-5 days by way of trypsinization and to test the cell viability the trypan blue method was used. The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0191] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0192] In T₀, 20 μl of MTS reagent was immediately added to the wells and they were read 90 minutes after. T₁ and T₂ were treated with different concentrations (2.5 μM to 25 μM) of L-733,060 and were incubated during a period of 30 hrs. (one cellular duplication) (T₁) and 72 hrs. (second cellular duplication) (T₂).

[0193] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by optical density, being directly proportional to the number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on a curve suited to the parameters.

[0194] Statistical Analysis: The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0195] Results: The results shown in FIG. 9A represent the variation in the time of the concentration of cells at growing concentrations of L-733,060.

[0196] In FIG. 9B the inhibition of cell growth MG-63 is represented (at 30 and 72 hrs.) after the addition of growing concentrations of L-733,060 (2.5, 5, 10, 20 and 25 μM). The percentage of the inhibition for the first and second time of the duplication of the incubation. The discontinuous lines represent the IC₅₀ at 30 and 72 hrs. The points on the graph represent the average value/typical deviation.

Example 11

Cell Lines Related to Glioma

[0197] A cell line related to human glioma GAMG (DSMZ) was used.

[0198] This cell line was maintained in a culture of MEN supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC.

[0199] The cell line was cultivated in 75 ml flasks (Falcon, Germany). Half were renewed every two days and the cells were treated with trypsin (0.05% and 0.02% EDTA without Ca²+ and Mg²+) every six days. The cells were incubated at a temperature of 37° C. in humidified (95% air/5% CO2).

[0200] Treatment with NK1 receptor antagonists: The solutions of the NK1 receptor antagonists (2S,3S)3-([3,5-Bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine, (L-733,060) (Sigma-Aldrich, U.K.) were dissolved in distilled water containing 0.2% dimethyl sulfoxide (DMSO) before treating the samples. Different concentrations of (2.5 μM to 25 μM) were studied to determine the IC₅₀.

[0201] The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the manufacturer's instructions for use (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA).

[0202] Method of Cellular Proliferation: During the experiment, the cultivated cells were broken apart every 4-5 days by way of trypsinization and to test the cell viability the trypan blue method was used. The cells were quantified and cultured in plates of 96 wells each. Each experiment included three plates termed T₀, T₁ and T₂.

[0203] T₀ contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10⁴ cells/0.1 ml) were termed control wells. Both T₁ and T₂ included white wells (0 cells/0.1 ml), control wells (10⁴ cells/0.1 ml) and control wells treated with L-733,060.

[0204] In T₀, 20 μl of MTS reagent was immediately added to the wells and they were read 90 minutes later. T₁ and T₂ were treated with different concentrations (2.5 μM to 25 μM) of L-733,060 and were incubated during a period of 48 hrs. (first cellular duplication) (T₁) and 96 hrs. (second cellular duplication) (T₂).

[0205] To study the proliferation of the cells, 20 μl of MTS reagent was added to each well (T₁, T₂) 90 min before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The quantity of MTS reagent was measured by optical density, being directly proportional in number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was performed in triplicate. The experiment was repeated on three different occasions. The concentration to inhibit fifty percent of the cells (IC₅₀) with L-733,060 was calculated on a curve suited to the parameters.

[0206] Statistical Analysis. The data obtained was evaluated using the Student's T Test, with a significance level of p<0.05.

[0207] Results: The results shown in FIG. 10A represent the variation in the time of the concentration of cells at growing concentrations of L-733,060.

[0208] In FIG. 10B the inhibition of cell growth GAMG is represented (at 48 and 96 hrs.) after the addition of growing concentrations of L-733,060 (10, 15, 20 and 25 μM). The percentage of the inhibition for the first and second time of the duplication of the incubation. The discontinuous lined represent the IC₅₀ at 30 and 72 hrs. The points on the graph represent the average value/typical deviation.

Claims

1. A pharmaceutical composition for the treatment of cancer in humans, comprising an effective amount of a non-peptide NK1 receptor antagonist, selected from the group consisting of aprepitant (MK 869 or L-754,030 (MSD)), L-758,298 (MSD), L-733,060 (MSD/Sigma-Aldrich), TAK-637 (Takeda/Abbot), GW597,599 (GSK), GW679,769 (GSK), and R673 (Roche), to achieve a concentration of between 5-50 μM at cancer cells to be treated.

2. The pharmaceutical composition according to claim 1, wherein the effective amount is sufficient to achieve a concentration of at least 20 μM.

3. A pharmaceutical composition for treating a human having cancel by inducing apoptosis in human cancer cells expressing at least 400% greater NK1 receptors than corresponding non-tumor cells of the same cell type, comprising an effective amount of a non-peptide NK1 receptor antagonist to achieve a concentration of at least an IC₅₀ for proliferation of the human cancer cells.

4. The pharmaceutical composition according to claim 3, wherein the effective amount is sufficient to achieve a concentration of at least 5 μM.

5. The pharmaceutical composition according to claim 3, wherein the effective amount is sufficient to achieve a concentration of at least 20 μM.

6. The pharmaceutical composition according to claim 3, comprising an effective amount of the non-peptide NK1 receptor antagonist to achieve a concentration of between about 5-50 μM.

7. The pharmaceutical composition according to claim 3, wherein the non-peptide NK1 receptor antagonist is selected from the group consisting of: L-733,060 (2S,3S) 3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine (MSD/Sigma-Aldrich); Vofopitant GR-205,171 (Pfizer); eziopitant CJ-11974 (Pfizer); CP-122,721 (Pfizer); Aprepitant (MK 869) (L-754,030) (MSD) (5-[[(2R,3S)-2-[(1R)-I-[3,5-bis(trifluoromethyl)phenyl]ethoxy]-3-(4-fluor- ophenyl)-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one); L-758,298 (MSD); TAK-637 (Takeda/Abbot); GW597,599 (GSK); GW679,769 (GSK); and R673 (Roche).

8. The pharmaceutical composition according to claim 7, wherein the non-peptide NK1 receptor antagonist comprises aprepitant.

9. The pharmaceutical composition according to claim 7, wherein the non-peptide NK1 receptor antagonist comprises L-733,060.

10. The pharmaceutical composition according to claim 7, wherein the non-peptide NK1 receptor antagonist comprises a 3,5-bis(trifluoromethyl)phenyl alkoxy ligand.

11. The pharmaceutical composition according to claim 7, wherein the non-peptide NK1 receptor antagonist comprises at least one trifluoromethyl ligand.

12. A pharmaceutical formulation for treatment of a tumor in an animal, the tumor having tumor cells which have a number of NK1 receptors of at least 400% of normal cells of the same type, comprising a non-peptide NK1 receptor antagonist in an amount sufficient to achieve a concentration of between about 5-50 μM in the animal.

13. The pharmaceutical formulation according to claim 12, comprising an effective amount of a non-peptide NK1 receptor antagonist to achieve a concentration of at least 20 μM.

14. The pharmaceutical formulation according to claim 12, wherein the non-peptide NK1 receptor antagonist is selected from the group consisting of: (2S,3S) 3-{[3,5-bis(trifluoromethyl)phenyl]methoxy}-2-phenylpiperidine L-733,060 (MSD/Sigma-Aldrich); Vofopitant GR-205171 (Pfizer); eziopitant CJ-11974 (Pfizer); CP-122721 (Pfizer); Aprepitant (MK 869) (L-754030) (MSD) (5-[[(2R,3S)-2-[(1R)-I-[3,5-bis(trifluoromethyl)phenyl]ethoxy]-3-(4-fluor- ophenyl)-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one); L-758298 (MSD); TAK-637 (Takeda/Abbot); GW597599 (GSK); GW679769 (GSK); and R673 (Roche).

15. The pharmaceutical formulation according to claim 14, wherein the non-peptide NK1 receptor antagonist comprises aprepitant.

16. The pharmaceutical formulation according to claim 14, wherein the non-peptide NK1 receptor antagonist comprises L-733,060.

17. The pharmaceutical formulation according to claim 14, wherein the non-peptide NK1 receptor antagonist comprises a 3,5-bis(trifluoromethyl)phenyl alkoxy ligand.

18. The pharmaceutical formulation according to claim 14, wherein the non-peptide NK1 receptor antagonist comprises at least one trifluoromethyl ligand.

19. The pharmaceutical formulation according to claim 12, wherein the amount sufficient to achieve a concentration of between about 5-50 μM in the animal is in excess of an IC₅₀ for proliferation of the tumor cells.

20. The pharmaceutical formulation according to claim 12, wherein the tumor cells are selected from the group consisting of: invasive primary and invasive malignant melanomas; metastatic melanoma cells; cells localized in ganglion lymph nodes; glioma cells--human breast cancer cells; Acute lymphoblastic leukemia B cells; Acute lymphoblastic leukemia T cells; primary neuroblastoma cells; astrocytoma cells; Burkitt's lymphoma cells; Hodgkin's lymphoma cells; Rhabdomyosarcoma cells; small lung cancer cells; Ewing's sarcoma cells; and osteosarcoma cells.

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