Abstract:
The invention relates to the use of substance P antagonists and, in particular, the use of non-peptidic NK1 receptor antagonists for the treatment of cancer and, more specifically, human melanoma, neuroblastoma, glioma, human Hodgkin&#39;s lymphoma KM-H2, lymphoblastic leukaemia, human rhabdomyosarcoma, human breast carcinoma, human Burkitt&#39;s lymphoma, human lung carcinoma, human Ewing&#39;s sarcoma, human glioma and human osteosarcoma.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     The present application is a 35 U.S.C. §371 national phase conversion of PCT/ES2005/000068, filed Feb. 10, 2005, which claims priority of Spanish Patent Application No. P200400424 filed Feb. 11, 2004, which is herein incorporated by reference. The PCT International Application was published in the Spanish language. 
     FIELD OF THE INVENTION 
     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&#39;s-KM-H2 lymphoma, lymphoblastic leukemia, Rhabdomyosarcoma, breast cancer, Burkitt&#39;s lymphoma, lung cancer, Edwing&#39;s sarcoma and human osteosarcoma. 
     BACKGROUND OF THE INVENTION 
     The P substance is a natural undecapeptide from the Tachykinins family and is used for its rapid stimulant action on smooth muscle tissue. More specifically, the P substance 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 the P substance, like in other Tachykinins, is seen in the physiopathology in a large number of illnesses that are well demonstrated in the bibliography. 
     The P substance receptor is a member of the super family of G-protein-coupled receptors. The neuropeptide receptor of the P (NK-1) substance 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. 
     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&#39;s disease. (Neurosci. Res., 1996, 7, 187-214), anxiety (Can. J. Phys.; 1997, 612-621) and depression (Science, 1998, 281, 1640-1645). 
     In the article titled “Tachykinin Receptor and Tachykinin Receptor Antagonists”, by J. Auton, in Pharmacol.; 1993, 13, 23-93, the use of antagonists of the P substance have been evidenced in the treatment of headaches, especially migraines, Alzheimer&#39;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. 
     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. 
     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. 
     The article in Life Sci.; 2000, 67(9), 985-1001 describes the Astrocytes express functional receptors for various neurotransmitters in the reception of the P substance. 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. 
     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. 
     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 the P substance (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 the 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). 
     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. 
     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 
     The objective of the current invention is the use of non-peptide NK1 receptor antagonists and the P substance 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. 
     The tumor cells that the antagonists act on are selected between 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&#39;s lymphoma cells; Hodgkin&#39;s lymphoma cells; Rhabdomyosarcoma cells; small lung cancer cells; Edwing&#39;s sarcoma cells; and osteosarcoma cells. They indicated the continuation in specific cells acted upon by the non-peptide NK1 receptor antagonists and the P substance. 
     The tumor cells related with human melanoma on which the current antagonists act in cellular lines are COLO 858 [ICLC, Interlab Cell Line Collection, CBA, Génova), MEL HO [DSMZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen] and COLO 679 [DSMZ]. 
     The tumor cells related with the human glioma and the human neuroblastoma to which the antagonists act on in cellular lines are the GAMG [DSMZ] and SKN-BE (2) [ICLC]. 
     The tumor cells related with 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 with Burkitt&#39;s lymphoma on which the antagonists act in cellular lines are CA-46 [DSMZ]. The tumor cells related with Hodgkin&#39;s lymphoma on which the antagonists act are KM-H2 [DSMZ]. The tumor cells related with rabdomio human sarcoma on which the antagonists act in a cellular linear form are A-204 [DSMZ]. The tumor cells related with small human lung cancer cells on which the antagonists act in a cellular line are COLO-677 [DSMZ]. The tumor cells related with human breast cancer on which the antagonists act in a cellular line are MT-3 [DSMZ]. 
     The tumor cells related with Edwing&#39;s sarcoma on which the current antagonists act in a cellular line are MHH-ES-1 [DSMZ]. 
     The tumor cells related with human osteosarcoma on which the current antagonists act in a cellular line are MG-63 [ICLC]. 
     One of the antagonists used is (2S,3S) 3-{[3,5-Bis(trifluoromethyl)phenyl]metoxi}-2-phenylpiperidine, commercially known as L-733060 (Sigma-Aldrich) and used in concentrations composed of between 5 suM and 50 I 1 M. 
     Other compounds of antagonist non-peptide receptors NK1 and the P substance that can be used include: vofopitant6GR-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). 
     Lastly, the other objective on the current invention is the use of the non-peptide NK1 receptor antagonists and the P substance, such as the aforementioned indicators in the creation of a pharmaceutical composition for the treatment of cancer. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIGS. 1A and 1B : variation in the time of the concentration of the cells SKN-BE (2) to growing concentrations of L-733,060( 1 A) in the cellular growth inhibition of SKN-BE (2)( 1  B). 
         FIGS. 2A and 2B : variation in the time of the concentration of the cells COLO 858 to growing concentrations of L-733,060( 2 A) in the cellular growth inhibition of COLO 858 ( 2 B). 
         FIGS. 3A and 3B : variation in the time of the concentration of the cells MEL HO to growing concentrations of L-733,060 ( 3 A) in the cellular growth inhibition of MEL HO ( 3 B). 
         FIGS. 4A and 4B : variation in the time of the concentration of the cells COLO 679 to growing concentrations of L-733,060 ( 4 A) in the cellular growth inhibition of COLO 679 ( 4 B). 
         FIG. 5 : variation in the time of the concentration of the cells SD1 to growing concentrations of L-733,060 in the cellular growth inhibition of SD1. 
         FIGS. 6A and 6B : variation in the time of the concentration of the cells KM-H2 to growing concentrations of L-733,060 ( 6 A) in the cellular growth inhibition of KM-H2 ( 6 B). 
         FIGS. 7A and 7B : variation in the time of the concentration of the cells MT-3 to growing concentrations of L-733,060 ( 7 A) in the cellular growth inhibition of MT-3 ( 7 B). 
         FIGS. 8A and 8B : variation in the time of the concentration of the cells MHH-ES-1 to growing concentrations of L-733,060 ( 8 A) in the cellular growth inhibition of MHH-ES-1 ( 8 B). 
         FIG. 9 : variation in the time of the concentration of the cells MG-63 to growing concentrations of L-733,060 in the cellular growth inhibition of MG-63. 
         FIGS. 10A and 10B : variation in the time of the concentration of the cells GAMG to growing concentrations of L-733,060 ( 10 A) in the cellular growth inhibition of GAMG ( 10 B). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A detailed explanation of how the activity was carried out was based on testing of aspects of the current invention in various cellular lines. The following examples are provided only in order to illustrate the invention and thus they should not be construed as limiting. 
     Example 1 
     Cellular lines related with neuroblastoma: Cellular lines of human neuroblastoma SKN-BE (2)(ICLC Interlab Cell Line Collection-CBA-Genova) was used. 
     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. 
     The cellular line was cultivated in flasks of 75 ml (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 a humidification of (95% air/5% CO2). 
     Treatment with the NK1 receptor antagonists: The solutions of antagonist NK1 receptors (2S,3S)3-([3,5Bis(trifluoromethyl)phenyl]metoxi)-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. 
     The proliferation of cells was tested using the MTS [3-(4,5-dimethylthiazol-2il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium]method, following the instructions established by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, (USA). 
     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 blue trypan 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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μl of MTS 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). 
     To study the proliferation of the cells 20 μl of MTS reactor was added to each well (T1,T2) 90 min., before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The reactive quantity of MTS, 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 done 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. 
     Statistical Analysis: The data obtained was evaluated using Student&#39;s t-test, with a significance level of P&lt;0.05. 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. 
     The  FIG. 1B  shows the 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) vs. the percentage of the inhibition in the first and second time in the duplication 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 
     Cellular lines related with melanomas Cellular lines related with melanomas COLO 858 (ICLC Interlab Cell LineCollection-CBA-Genova), MEL HO and COLO 679 (DSMZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen) were used. 
     This cellular 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, the ICLC and the DSMZ. 
     The cellular lines were cultivated in flasks of 75 ml (Falcon, Germany). 
     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 a humidification of (95% air/5% CO2). 
     The treatment with NK7 receptor antagonists (2S,3S) 3-([3,5 Bis(trifluoromethyl)phenyl]metoxi)-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. 
     The cellular proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], according to the instructions of use established by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     Cellular Deproliferation 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 blue trypan 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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     20 μl of MTS 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 cellular lines. 
     Line COLO 858: 48 h. (first cellular duplication) (T1) and 96 hrs. (second cellular duplication) (T2). 
     Line MEL HO: 24 hrs. (cellular duplication) (T1) and 48 hrs. (second cellular duplication) (T2). 
     Line COLO 679: 30 hrs. (cellular duplication) (T1) and 72 hrs. (second cellular duplication)(T2). 
     To study the cellular proliferation, 20 μl of reactive MTS was added to each well (T1, T2) 90 min. before reading the plate samples with the (TECAN Spectra Classic) 492 nm. the quantity of reactive MTS, the optical density was measured, being directly proportional to the number of live cells. Each plate (white, control, and control treated with different concentrations of L-733,060) was done 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. 
     Statistical Analysis: The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     Results: The results are shown in  FIGS. 2A, 2B  (COLO 858),  FIGS. 3A and 3B  (MEL HO) and  FIGS. 4A and 4B  (COLO 679). 
       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). 
       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). 
       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). 
     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. 
     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. 
     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 
     Cellular lines related with lymphoblastic leukemia. Cellular lines related with human lymphoblastic leukemia have been used with BSD1 (DSMZ) and the cells T BE-13 (DSMZ). 
     These cellular lines were maintained in a culture of 1640 supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC. 
     The cellular line was cultivated in flasks of 75 ml (Falcon, Germany). Half were renewed every two days. The cells were incubated at a temperature of 37 C in a humidification of (95% air/5% CO2). 
     Treatment with Antagonist NK1 Receptors 
     The solutions of the antagonist NK1 receptors (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]metoxi)-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 at 25 μM) were studied in order to determine the IC50. 
     The cellular proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μl of MTS 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. (cellular duplication) (T1) and 72 hrs. (second cellular duplication)(T2). 
     To study the proliferation of the cells 20 μl of reactive MTS 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 reactive MTS, 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 done 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. 
     Statistical Analysis: The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     Results: The results shown in  FIG. 5  represents the variation in time of the concentration of cells B SD1 to growing concentrations of L-733,060. 
     Example 4 
     Cellular lines related with Burkitt&#39;s human lymphoma The cellular line of Burkitt&#39;s human lymphoma was used with CA-46 (DSMZ). 
     This cellular 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. 
     The cellular lines were cultivated in flasks of 75 ml (Falcon, Germany). Half was renewed every two days and the cells were treated with Trypsin (0.05% and 0.02% EDTA in Ca2+ and Mg2+) every six days. The cells were incubated at 37 C in a humidification of (95% air/5% CO2). 
     Treatment with NK1 receptor antagonists The solutions of the NK1 receptor antagonists (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]metoxi)-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 IC50. 
     The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     The discontinuous lines represent the IC50 at 30 and 72 hrs. The points on the graph show the average value/typical deviation. 
     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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μL of MTS 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). 
     To study the proliferation of the cells 20 pi of reactive MTS 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 reactive MTS 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 done 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. 
     Statistical Analysis: The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     Results: At the highest concentrations, inhibition in cellular growth was produced and at the maximum dose, apoptosis. 
     Example 5 
     Cellular lines related with human Hodgkin&#39;s lymphoma. Cellular lines related with human Hodgkin&#39;s lymphoma. KM-H2 (DSMZ) were used. 
     This cellular 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. 
     The cellular line was cultivated in flasks of 75 ml (Falcon, Germany). Half were renewed every two days. The cells were incubated at a temperature of 37 C in a humidification of (95% air/5% CO2). 
     Treatment with NK1 receptor antagonists The solutions of the NK1 receptor antagonists (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]metoxi)-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 IC50. 
     The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells./0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μl of MTS 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 48 hrs. (cellular duplication) (T1) and 96 hrs. (second cellular duplication)(T2). 
     To study the proliferation of the cells 20 μL of reactive MTS 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 reactive MTS, 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 done 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. 
     The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     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. 
     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 
     Cellular Lines Related to Human Rhabdomyosarcoma. 
     Cellular Lines Related to Human Rhabdomyosarcoma A-204 (DSMZ) were Used. 
     This cellular 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. 
     The cellular line was cultivated in flasks of 75 ml (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 a humidification of (95% air/5% CO2). 
     Treatment with antagonist NK1 receptors The solutions of the antagonist NK1 receptors (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]metoxi)-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 IC50. 
     The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     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 blue trypan 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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μl of MTS 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). 
     To study the proliferation of the cells 20 μl of MTS reactor was added to each well (T1, T2) 90 min., before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The reactive quantity of MTS, 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 done 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. 
     Statistical Analysis: The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     Results: Cellular growth is inhibited at the highest concentrations and at the maximum dose, apoptosis. 
     Example 7 
     Cellular Lines Related with Small Cell Lung Cancer 
     Cellular lines related with small cell lung cancer COLO-677 (DSMZ) was used. This cellular 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. 
     The cellular line was cultivated in flasks of 75 ml (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 a humidification of (95% air/5% CO2). 
     Treatment with antagonist NK1 receptors The solutions of the antagonist NK1 receptors (2S,3S) 3-[(3,5-Bis(trifluoromethyl)phenyl]metoxi)-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. 
     The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     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 blue trypan 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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μl of MTS 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). 
     To study the proliferation of the cells 20 μL of MTS reactor was added to each well (T1, T2) 90 min., before reading the samples with the plate reader (TECAN Spectra Classic) at 492 nm. The reactive quantity of MTS, 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 done 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. 
     Statistical Analysis: The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. Results: Cellular growth is inhibited at the highest concentrations and at the maximum dose, apoptosis. 
     Example 8 
     Cellular lines related with human breast cancer Cellular lines related with human breast cancer MT-3 (DSMZ) was used. This cellular 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. 
     The cellular line was cultivated in flasks of 75 ml (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 a humidification of (95% air/5% CO2). 
     Treatment with NK1 receptor antagonists The solutions of the NK1 receptor antagonists (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]metoxi)-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. 
     The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     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 blue trypan 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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μl of MTS 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). 
     To study the proliferation of the cells 20 pi of reactive MTS 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 reactive MTS, 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 done 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. 
     Statistical Analysis: The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     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. 
     In  FIG. 7B  the inhibition of cell growth MT-3 is represented (at 30 and 72 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 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 
     Cellular lines related to Edwing&#39;s human sarcoma Cellular lines related to Edwing&#39;s human sarcoma MHH-ES-1 (DSMZ) were used. This cellular 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. 
     The cellular line was cultivated in flasks of 75 ml (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 a humidification of (95% air/5% CO2). 
     Treatment with NK1 receptor antagonists The solutions of the NK1 receptor antagonists (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]metoxi)-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. 
     The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     Method of Cellular Proliferation 
     During the experiment, the cultivated cells were broken apart every 4-5 days by way of tripsinization and to test the cell viability the blue trypan 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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells./0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 PI of MTS 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). 
     To study the proliferation of the cells 20 μl of reactive MTS 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 reactive MTS, 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 done 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. 
     Statistical Analysis: The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     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. 
     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 
     Cellular line related to human osteosarcoma Cellular lines related to human osteosarcoma MG-63 (ICLC) were used. 
     This cellular line was maintained in a culture of MEN supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC. 
     The cellular line was cultivated in flasks of 75 ml (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 a humidification of (95% air/5% CO2). 
     Treatment with NK1 receptor antagonists The solutions of the NK1 receptor antagonists (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]metoxi)-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. 
     The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     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 blue trypan 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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μl of MTS 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) 
     To study the proliferation of the cells 20 μl of reactive MTS 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 reactive MTS, 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 done 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. 
     Statistical Analysis: The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     Results: The results shown in  FIG. 9  represent the variation in the time of the concentration of cells at growing concentrations of L-733,060. 
     Example 11 
     Cellular lines related to glioma Cellular lines related to human glioma GAMG (DSMZ) was used. 
     This cellular line was maintained in a culture of MEN supplemented with 10% fetal bovine serum according to the established cellular culture conditions of the ATCC. 
     The cellular line was cultivated in flasks of 75 ml (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 a humidification of (95% air/5% CO2). 
     Treatment with NK1 receptor antagonists The solutions of the NK1 receptor antagonists (2S,3S) 3-([3,5-Bis(trifluoromethyl)phenyl]metoxi)-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. 
     The cell proliferation was evaluated using the MTS method [3-(4,5-dimethylthiazol-2-il)-5-(3-carboxymethoxyphenyl) 2-(4-sulfophenyl)-2H-tetrazolium], following the instructions of use by the manufacturer (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, USA). 
     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 blue trypan 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. 
     T0 contained wells without cells (0 cells/0.1 ml) termed white wells and wells that contained cells (10 4  cells/0.1 ml) were termed control wells. Both, T1 and T2, included white wells (0 cells/0.1 ml), control wells (10 4  cells/0.1 ml) and control wells treated with L-733,060. 
     In T0, 20 μl of MTS 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). 
     To study the proliferation of the cells 20 μl of reactive MTS 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 reactive MTS, 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 done 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. 
     Statistical Analysis. The data obtained was evaluated using the Student&#39;s t-test, with a significance level of P&lt;0.05. 
     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. 
     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 lines represent the IC50 at 30 and 72 hrs. The points on the graph represent the average value/typical deviation.