KR101847217B1 - Methods for providing personalized medicine tests ex vivo for hematological neoplasms - Google Patents

Abstract

Disclosed are methods, devices, and compositions for providing customized medical examinations for serological neoplasms. In certain embodiments, the method comprises measuring the efficacy of selectively inducing apoptosis in malignant cells using any number of potential alternative drug combinations. In certain embodiments, in vitro tests are performed using recently extracted patient serological samples. In another embodiment, the efficacy is measured in vitro using automated flow cytometry. For example, by using an automated pharmacokinetic assay platform, the evaluation of hundreds or even thousands of drugs and compositions can be performed in vitro. Thus, alternative multifunctional therapies can be studied. Non-cytotoxic drugs surprisingly induce apoptosis selectively in in vitro malignant cells. In certain embodiments, the methods comprise evaluating non-cytotoxic drugs.

Description

[0001] METHOD FOR PROVIDING IN VIVO FOR NEUTRAL NEOPLASMS [0002]

The present invention relates to the use of screening flats to determine the cytotoxic drug sensitivity properties (cytotoxic drug sensitivity properties) of multiple drug and drug combinations using a sample of cancer patients.

There are many methods available for evaluating the cytotoxic drug susceptibility properties of tumor cells in in vitro samples from cancer patients. In vitro assays for detecting apoptosis in serologic neoplasms have been developed over the past 40 years and as a result, numerous assays have been proposed to confirm chemosensitivity. The Individualized Tumor Response Test / Testing (ITRT) has recently been proposed to describe such an approach to describe the "effect of chemotherapy on fresh whole biopsied tumor cells from cancer patients" (Bosanquet et al. GJ Kaspers, B. Coiffier, MC Heinrich and EH Estey, New York, NY, 2008, Informa Healthcare: 23-44). ITRTs (eg, clonogenic assays) designed to study the ability of drugs to slow or stop the growth of new cells did not initially work well. However, in the 1980s, a number of ITRTs were developed for apoptosis, which showed a good comparable agreement between analysis results and clinical outcomes (eg, clinical correlations).

While there are good clinical correlations, currently available ITRTs for apoptosis have undesirable limitations that limit their use as customized medical tests. For example, colony formation assays limit their clinical usefulness by requiring weeks rather than days to derive results (Hamburger et al., Science 1911, 197: 461-463; Marie et al., Br J Haematol 1983 , 55: 427-437; Selby et al., New Engl J Med 1983,308: 129-134). In addition, most ITRTs measure total cell death to assess the effects of drug and in vitro application of samples. Measuring total cell death limits the ability of ITRT to distinguish the effect of drugs on tumor cells against normal cells. Currently available ITRTs differ mainly from the methodology used to determine the proportion of live cells or dead tumor cells at the end of the assay.

Some ITRTs measure cells directly, but most are using indirect markers to assess cell death indirectly. For example, the MTT (methyl-thiazolyltetrazolium) assay is to estimate the number of viable cells by deriving mitochondrial reduction of MTT to formazan with a quantifiable color change and measuring it with a spectrophotometer (Pieters Blood 1990, 76: 2327-2336; Sargent et al., Br J Cancer 1989, 60: 206-210; Carmichael et al., Cancer Res 1987, 47: 936-942). Other ITRTs utilize the amount of cellular ATP, either by using fluorescein diacetate hydrolysis (e.g., fluorometric microculture cytotoxicity assay (FMCA)) or indirect markers of cellular activity (Rhedin et al., Leuk Res (Differential Staining Cytotoxicity) assays and more recent TRACs (tumor response to anti-neoplastic compounds (see, for example, Jensen et al., 1993, 17: 271-276; Larsson et al., Int J Cancer 1992, 50: 177-185) Tumor Response to Antineoplastic Compounds) determine the viable tumor cells by microscopy using the staining method (Bosanquet et al., Br J Haematol 2009, 146: 384-395; Bosanquet et al., Leuk Res 1996, 20: 143-153; Weisenthal Cancer Res 1983, 43: 749-757).

The aforementioned ITRTs require culturing the patient's neoplastic cells and cytotoxic drugs for at least 4 to 5 days. However, serologic cells begin to lose important traits after 24 to 48 hours outside the human body. The shorter incubation will allow the in vitro cytotoxicity properties to be assessed prior to the start of patient treatment, thereby increasing their clinical usefulness and enabling more effective application as a customized medical examination.

Cytotoxic drugs appear to eliminate malignant cells by inducing apoptosis (Aragane et al., / Cell Biol 1998, 140: 171-182; Hannun et al., Blood 1997, 89: 1845-1853). Apoptosis is a type of apoptosis, commonly referred to in the art as " programmed apoptosis " and is defined according to morphological and antigenic characteristics. Apoptosis usually begins within a few hours after the drug contacts the target cell (Del Bino et al., Cell Prolif 1999, 32: 25-37). There are many apoptosis assays based on markers that reflect different aspects of the apoptosis process. For example: 1) Changes in mitochondrial potential membrane using DiOC6 or JC-I (Tabrizi et al., Leukemia 2002, 16: 1154-1159; Liu et al., Leukemia 2002, 16: 223-232); 2) Chromosomal DNA fractions were identified by Tdt and electrophoresis was performed using the terminal deoxynucleotidyl transferase (TUNEL) assay (Liu et al., Leukemia 2002, 16: 223-232) Cancer Res Clin Oncol 2002, 128: 375-377 (1999)), or by acridine orange (Tabrizi et al., Leukemia 2002, 16 (6): 1154-9; Kim et al., Exp MoI Med 2000, 32: 197-203; Konstantinov et al. Ofir et al., Cell Death Differ 2002, 9: 636-642); Or 3) identifying a proteolytic cleavage of poly-ADP-ribose polymerase (PARP) or caspase-3 using a specific antibody (Konstantinov et al. / Cancer Res Clin Oncol 2002i 128 (5): 271-8 Oncogene 2004, 23: 4523-4535, Prokop et al., Oncogene 2003, 22: 4523-4535, 9107-9120).

Another assay for apoptosis is based on the detection of annexin V bound to a fluorescent marker (e.g., fluorochrome) by flow cytometry. Annexin V binds to externalized phosphatidylserine residues that appear only on the surface membrane of cells undergoing apoptosis (Tabrizi et al., 2002; 16 (6): 1154-9; Nimmanapalli et al., Cancer Res 2002, 62 : 5761-5769). Measurement of apoptosis can be determined by the ratio of cells that are associated with annexin V-fluorescent complex upon detection by flow cytometry. In addition, several monoclonal antibody combinations commonly used in the identification of tumor cells (normal cells) are known in the art. Table 1 shows various monoclonal antibodies that, when combined with fluorochrome, can identify serologic tumor cells using various spectroscopic detection methods.

<Monoclonal antibody combination for tumor cell identification> Serological neoplasm AcM-hemochromic dye conjugate ALL, CLL, NHL CD19-PE, CD45-APC MM CD38-PE, CD45-APC AML CD34-PE, CD45-APC

ALL = acute lymphocytic leukemia; CLL = chronic lymphocytic leukemia; NHL = non-Hodgkin lymphoma; MM = multiple myeloma; AML = acute myelogenous leukemia

Certain ITRTs, particularly the DiSC and TRAC assays, can simultaneously measure cytotoxicity in tumor cells and normal cells, thus determining therapeutic indices (Bosanquet et al., Leuk. Res. 1996; 20: 143-53; Bosanquet J Exp Ther Oncol 2004; 4: 145-54).

The researchers have suggested the ability of ITRTs to predict in some scientific reviews. A review of 1929 clinical correlations in serologic malignancies (Bosanquet et al., In Kaspers et al., (2008)) and other reviews (eg, Kaspers GJ., Methods MoI Med. 2005; 110: 49-57), especially in terms of drug tolerance, positive predictive efficiency appears at a high rate. Nagourney found that the positive predictive efficiency for drug sensitivity was 81.8% and the negative predictive efficiency for drug resistance was 83.3% (Source: http: //www.rationaltherapeutics.com/physicians/contentl.aspx? rid = 35 and bibliographic references here (visit May 7, 2010)).

<List of published clinical correlates supporting the predictive power of the ITRT> Hematologic cancer N TP TN FP FN Ref. ALL 3 2 One 0 0 One ALL 17 14 2 One 0 2 ALL 25 16 3 5 One 3 ALL 130 90 18 20 2 4 ALL 58 40 6 0 12 5 ALL 4 One 2 One 0 6 ALL 4 3 One 0 0 7 ALL 29 18 5 2 4 8 ALL 2 2 0 0 0 9 ALL / CLL 55 38 7 10 0 10 AML 4 0 One 2 One 2 AML 11 6 5 0 0 11 AML 21 11 8 2 0 6 AML 83 74 9 0 0 12 AML 27 6 13 0 8 13 AML 21 10 9 2 0 14 AML 33 11 8 4 10 15 AML / ALL / NHL 73 45 16 9 3 16 AML 12 7 3 2 0 17 AML 14 9 One 2 2 3 AML 14 9 2 One 2 4 AML 17 11 4 One One 7 AML 27 12 12 2 One 18 AML 34 20 11 2 One 19 CLL 80 12 48 18 2 2 CLL 34 26 6 2 0 20 CLL One One 0 0 0 6 CLL 15 11 3 0 One 21 CLL 15 9 4 One One 8 CLL 3 2 One 0 0 9 CLL / ALL / NHL 226 102 76 41 7 22 CLL 9 2 6 One 0 8 NHL One One 0 0 0 17 NHL 10 3 3 3 One 2 NHL 3 2 0 One 0 One NHL 50 27 10 11 2 23 NHL 10 6 3 One 0 8 NHL 3 0 3 0 0 9 sum 1178 659 310 147 62

N = number of events; TP = true positive; TN = true voice; FP = spurious positive; FN = spurious voice; Ref. = Bibliography (reference at the end of the reference specification); ALL = acute lymphocytic leukemia; CLL = chronic lymphocytic leukemia; AML = acute myeloid leukemia; NHL = non-Hodgkin lymphoma

 A prospective randomized controlled clinical trial is currently being conducted in the UK using a large number of patients with chronic lymphocytic leukemia (UK LRF CLL4 trial, Catovsky et al., Lancet 2007, 370: 230-39). This study is to evaluate the ITRT as a performance factor related to patient response to therapy (Bosanquet et al., ASH Annual Meeting Abstracts Blood, 2006 108: 94a: Abstract 303). This trial began in 1999 and includes 777 previously untreated CLL patients. Patients were treated with chlorambucil (ChI) or fluaravarin +/- cyclophosphamide (Flu or FIuCy). In this study, TRAC assays were used to assess the in vitro susceptibility to drugs before patient treatment. For analysis, patients were divided into three groups according to the results of the ITRT: drug resistance (DR), drug susceptibility (DS), or drug intermediate group (DI). Table 3 summarizes these results.

Correlation between ITRT results (DS, DI, and DiL) and response to the same medication in patients with chronic lymphocytic leukemia> Result ChI Flu FIuCy Total DS 85.1 (94) 90.7 (54) 95.7 (70) 89.9 (218) DI 66.3 (92) 79.2 (53) 97.3 (37) 76.4 (182) DR 37.6 (24) 21.4 (14) 25.0 (4) 31.0 (42) Total 71.5 (210) 77.7 (121) 93.7 (111) 78.7 (442)

Results are expressed as a percentage of patients (number of patients in parentheses) who responded to each drug (ChI and Flu) and drug combination (FIuCy).

 As shown in Table 3, ITRT results are well correlated with patient clinical responses. Of the 49% of DS patients, most (90%) responded to chemotherapy, whereas only 9% of patients with DR responded to chemotherapy. Among 24 patients with DR in ChI, 71% were DS or DI in Flu and all were DS or DI for FIuCy combination. Of the 14 patients with DR in Flu, only 36% were DS or DI for the FluCy combination. These results suggest that using ITRT results may lead to more efficient treatment and better clinical outcomes.

 If there is a tremendous therapeutic potential of customized medical testing, there is a need to develop an ITRT that uses shorter incubation times in the art. The use of such assays to assist in the selection of therapies may significantly increase the rate of response, progression-free survival time and overall survival time of cancer patients. Preferably, the assay allows for the measurement of individual tumor cell death using flow cytometry and reduces the assay time to obtain cytotoxic properties in a short time. Also preferred is an ITRT that provides a concentration of a number of pharmaceuticals and agents that are effective, either individually or in combination

It is an object of the present invention to provide a method for providing an in vitro customized medical examination for serological neoplasia.

 The present invention relates to the development of customized medical tests for patients. In a general embodiment, the invention relates to compositions, methods and systems for analyzing cellular responses to drugs using in vitro assays. What is presented in the present invention is a method of analyzing a whole blood sample by dealing with a number of variables and quickly completing the analysis.

 In one embodiment, there is provided a method of analyzing cellular response to a drug, comprising: obtaining a sample of tissue from a serological neoplasm obtained from a patient; Dividing the sample of tissue into at least 35 aliquots; Combining said at least 35 aliquots each with a drug composition; And measuring apoptosis in at least one cell population in each of said at least 35 aliquots. In one embodiment, the tissue obtained from the serological neoplasm is a tissue selected from the group consisting of peripheral blood, bone marrow, lymph nodes, and spleen. In another embodiment, the sample is frozen or cryopreserved, and the frozen or cryopreserved sample is thawed before being divided into at least 35 aliquots. In another embodiment, the measuring step is completed within 72 hours of combining the aliquot with the drug composition. In another embodiment, the measuring step is completed within 48 hours of combining the aliquot with the drug composition. In another embodiment, the measuring step is completed within 24 hours of combining the aliquot with the drug composition. In another embodiment, the measuring step is performed using a flow cytometer. In other embodiments, the number of aliquots having a unique drug composition is at least 96. In another embodiment, at least two of the drug compositions comprise the same drug at different concentrations. In another embodiment, at least one of the drug compositions comprises a plurality of drugs. In another embodiment, at least one of the drug compositions comprises a plurality of drugs that are non-cytotoxic. In another embodiment, at least one of the drug compositions comprises a non-cytotoxic drug of the same species or in the same therapeutic field as the drug already administered to the patient. In another embodiment, at least one of the drug compositions is a combination of a non-cytotoxic drug and a cytotoxic drug. In other embodiments, the apoptosis is selectively measured for a particular population of cells. In another embodiment, the Apoptosis is measured against a population of cells representing the serological neoplasm. In another embodiment, the serological neoplasm is selected from the group consisting of: chronic lymphocytic leukemia, adult acute lymphocytic leukemia, pediatric acute lymphocytic leukemia, multiple myeloma, myelodysplastic syndrome, non-M3 acute myeloid leukemia, acute myelogenous leukemia M3, non-Hodgkin lymphoma, Hodgkin's lymphoma, and chronic myelogenous leukemia. In another embodiment, at least one of the drug compositions comprises a combination of fludarabine or chlorambucil with sertraline, paroxetine, or fluoxetine. In another embodiment, at least one of the drug compositions comprises fludarabine and a cyclophosphamide. In another embodiment, the method comprises injecting a mouse into a cell obtained from a sample of tissue obtained from a serological neoplasm; Allowing the injected cells to proliferate in the mice for a sufficient time; And removing the proliferated cells from the mice, wherein the infusion, proliferation and elimination are performed prior to combining the fractions with the drug composition. In another embodiment, the method includes generating a report that summarizes the results of the measurement step. In another embodiment, the method further comprises providing the report to a person concerned with the medical care of the patient. In another embodiment, the drug composition is selected from the group consisting of 5-azacytidine, alemtuzumab, aminopterin, amonafide, amsacrine, CAT-8015, bevacizumab, ARR Y520, arsenic trioxide, AS1413, ATRA ), AZD 6244, AZDl 152, vanoxanthrone, behenoylana-C, vendamustine, bleomycin, blana tomomab, bortezomib, overlaid, carboplatin, CEP-701, chlorambucil, Lysine-A &lt; / RTI &gt; chain, &lt; RTI ID = 0.0 &gt; lysine-A &lt; / RTI &gt; chain - conjugated anti-CD 19 / anti-CD22 immunotoxins, dexamethasone, doxorubicin, elastase rabine, entinostat, fructus jum, euinase, etofoside, everonimus, exatexan mesylate, Stones, fludarabine, porodensin, gemcitabine, gemtuzumab - ozogamicin, homo-marine tonin, Isoindine I 131 monoclonal antibody BC8, isospetemide, isotretinoin, raromustine, L-asparaginase, lenalidomide, lidocaine, hydrocortisone, hydroxycarbamide, dirubicin, But are not limited to, methotrexate, methylprednisolone, methylprednisone, middysterin, mitoxantrone, nelalabine, neurotinib, oblimersen, paclitaxel, panovinostat, Paparestin, pyrabicin, PKC412, prednisolone, prednisone, PSC-833, rapamycin, rituximab, ribavirin, sapacitabine, dinacilip, sorafenib, sorafenib, STA-9090 , Tacrolimus, tanepsimycin, temsirolimus, tennyfoside, terameprocole, thalidomide, thioguanine, thiotepa, tififanib, topotecan, thiosulfan, Binh, Vincristine, Vincristine, Vindeshin, Vinoreveline, Borrexin, Borinostat, Etoposide, Tidal Quidar. In another embodiment, the pharmaceutical composition is selected from the group consisting of aluminum oxide hydrate, lorazepam, amikacin, meropenem, cefepime, vancomycin, teicoplanin, ondansetron, dexamethasone, amphotericin B (liposomal) , Selected from the group consisting of itraconazole, fluconazole, voriconazole, trimethoprim, sulfamethoxazole, G-CSF, ranitidine, rasburicase, paracetamol, metamizole, morpholine chloride, omeprazole, paroxetine, fluoxetine, sertraline &Lt; / RTI &gt;

 In another embodiment, there is provided a method of analyzing the response of a neoplastic cell to a drug, comprising: obtaining a sample of tissue from a serological neoplasm collected from a patient; Separating the sample of tissue into at least 35 aliquots; Wherein the at least one of the at least 35 fractions is combined with the drug composition, wherein the drug composition in each aliquot differs in at least one of the identity, concentration, or combination thereof of the drug composition and the drug in all the other fractions, Wherein the drug compositions collectively comprise at least one non-cytotoxic drug; Culturing said fraction in combination with a drug composition; And analyzing, for each cultured aliquot, the response of at least one type of neoplastic cell to the drug composition. In one embodiment, the tissue is selected from the group consisting of peripheral blood, bone marrow, lymph nodes, and spleen. In another embodiment, the sample is a frozen or cryopreserved sample, and the frozen or cryopreserved sample is thawed prior to dividing the sample into at least 35 aliquots. In another embodiment, the analyzing step is completed within 72 hours of combining the aliquots with the drug composition. In another embodiment, the analysis step is completed within 48 hours of combining the aliquots with the drug composition. In another embodiment, the analysis step is completed within 24 hours of combining the aliquots with the drug composition. In another embodiment, the method further comprises creating a report that summarizes the results of the analysis step. In another embodiment, the method further comprises providing the report to a person concerned with the medical care of the patient. In another embodiment, the number of aliquots combined with the drug is at least about 96. In another embodiment, the measuring step is performed using a flow cytometer. In another embodiment, the neoplastic cell is a serologic neoplasm. In another embodiment, the serologic neoplasms include: chronic lymphocytic leukemia, adult acute lymphocytic leukemia, childhood acute lymphocytic leukemia multiple myeloma, myelodysplastic syndrome, non-M3 acute myelogenous leukemia, acute myelogenous leukemia M3, Non-Hodgkin's lymphoma, Hodgkin's lymphoma, and chronic myelogenous leukemia. In another embodiment, the method comprises injecting a mouse with a neoplastic cell obtained from a sample of tissue; Allowing the injected neoplastic cell to proliferate in the mouse for a sufficient time; And removing the proliferated neoplastic cell from the mouse, wherein the infusion, proliferation, and elimination steps are performed prior to combining the fraction with the drug composition. In another embodiment, the drug composition is selected from the group consisting of 5-azacytidine, alemtuzumab, aminopterin, amonafide, amsacrine, CAT-8015, bevacizumab, ARR Y520, arsenic trioxide, AS1413, ATRA ), AZD 6244, AZDl 152, vanoxanthrone, behenoylana-C, vendamustine, bleomycin, blana tomomab, bortezomib, overlaid, carboplatin, CEP-701, chlorambucil, Lysine-A &lt; / RTI &gt; chain, &lt; RTI ID = 0.0 &gt; lysine-A &lt; / RTI &gt; chain - conjugated anti-CD 19 / anti-CD22 immunotoxins, dexamethasone, doxorubicin, elastase rabine, entinostat, fructus jum, euinase, etofoside, everonimus, exatexan mesylate, Stones, fludarabine, porodensin, gemcitabine, gemtuzumab - ozogamicin, homo-marine tonin, Isoindine I 131 monoclonal antibody BC8, isospetemide, isotretinoin, raromustine, L-asparaginase, lenalidomide, lidocaine, hydrocortisone, hydroxycarbamide, dirubicin, But are not limited to, methotrexate, methylprednisolone, methylprednisone, middysterin, mitoxantrone, nelalabine, neurotinib, oblimersen, paclitaxel, panovinostat, Paparestin, pyrabicin, PKC412, prednisolone, prednisone, PSC-833, rapamycin, rituximab, ribavirin, sapacitabine, dinacilip, sorafenib, sorafenib, STA-9090 , Tacrolimus, tanepsimycin, temsirolimus, tennyfoside, terameprocole, thalidomide, thioguanine, thiotepa, tififanib, topotecan, thiosulfan, A compound selected from the group consisting of blanch, vinblastine, vincristine, vindesine, vinorelbine, borreoxine, borinostat, etoposide, anhydrous quidar. In another embodiment, the pharmaceutical composition is selected from the group consisting of aluminum oxide hydrate, lorazepam, amikacin, meropenem, cefepime, vancomycin, teicoplanin, ondansetron, dexamethasone, amphotericin B (liposomal) , Selected from the group consisting of itraconazole, fluconazole, voriconazole, trimethoprim, sulfamethoxazole, G-CSF, ranitidine, rasburicase, paracetamol, metamizole, morpholine chloride, omeprazole, paroxetine, fluoxetine, sertraline &Lt; / RTI &gt;

 In another embodiment, there is provided a method of facilitating the treatment of serological neoplasia in a patient, comprising: providing a tissue sample obtained from a patient, comprising a neoplastic cell; Culturing each of at least six portions of the sample with a different drug or drug combination; Analyzing each of the portions of the sample to determine the extent of apoptosis of the neoplastic cells in the portion; And issuing a printed or electronic report of the results from the analysis step, which indicates at least a portion, drug or drug combination having maximal apoptosis. In one embodiment, the resulting report shows results from a plurality of drugs or drug combinations. In other embodiments, the analysis and incubation steps further comprise additional portions that differ in drug concentration from the other portions.

 In another embodiment, a plurality of chambers; An apparatus for analyzing a response of a neoplastic cell to a potential drug regimen, wherein the chamber comprises collectively: at least one chamber comprising a plurality of drugs; At least one chamber comprising a cytotoxic drug; And at least a total of 10 different drugs in the collective chamber. In one embodiment, the device comprises at least one chamber comprising a non-cytotoxic drug. In another embodiment, the device further comprises at least one chamber comprising a cytotoxic drug and a non-cytotoxic drug. In another embodiment, the device further comprises at least two chambers containing the same drug at different concentrations. In other embodiments, at least one of the chambers comprises fluaravarin or chlorambucil in combination with sertraline, paroxetine, or fluoxetine. In another embodiment, at least one of the chambers comprises fluaravarin and a cyclophosphamide. In another embodiment, at least one of the at least 10 different drug compositions is selected from the group consisting of 5-azacytidine, alemtuzumab, aminopterin, amonafide, amsacrine, CAT-8015, bevacizumab, ARR Y520, arsenic trioxide, CEP-701, Carboprolactin, CEP-701, Bacillus amyloliquefaciens, ATRA, AZD 6244, AZDl 152, vanoxanthrone, behenoylana-C, vendamustine, Clorribine, Clofarabine, CPX-351, Cyclophosphamide, Cyclosporin, Cytarabine, Cytosine arabinoside, Dasatinib, Daunorubicin, Decitabine, Fused-lysine-A chain-conjugated anti-CD19 / anti-CD22 immunotoxins, dexamethasone, doxorubicin, elastase rabine, entinostat, fructus jum, euinase, etoposide, everonimus, Candesartan, candesartan, candesartan, candemisilate, flavopiridol, fludarabine, porodysin, gemcitabine, Isoindymine, Iodine I 131 131 Monoclonal antibody BC8, Ipsospimide, Isotretinoin, Lauro Muram - Ozogamicin, Homo Haringtonin, Hydrocortisone, Hydroxycarbamide, Dirubicin, Iphospamide, Imatinib, Interferon alfa 2a, But are not limited to, cholinesterase inhibitors such as L-asparaginase, L-asparaginase, lenalidomide, lestaurtinib, mapospamide, melphalan, mercapturidine, methotrexate, methylprednisolone, methylprednisone, midosutaurin, mitoxantrone, Paclitaxel, pravastatin, pravastatin, firarubicin, PKC412, prednisolone, prednisone, PSC-833, rapamycin, rituximab, ribavirin, , Sorafenib, sorapenib, STA-9090, tacrolimus, tanemspimycin, temsirolimus, tennyfoside, terameprocol, thalidomide, thioguanine, thiotepa, Is selected from Tecan, threo 0498, teurok perspective turbine, vinblastine, vincristine, binde new, unexposed mozzarella bar, Borel roksin, barley furnace stats, Ⅰ Topo side, and the group consisting of fresh water in the different rake. In another embodiment, one or more of the 10 different drug compositions are selected from the group consisting of aluminum oxide hydrate, lorazepam, amikacin, meropenem, cefepime, vancomycin, teicoplanin, ondansetron, dexamethasone, amphotericin B Liposomal), carpartofugine, itraconazole, fluconazole, voriconazole, trimethoprim, sulfamethoxazole, G-CSF, ranitidine, rasburicase, paracetamol, metamizole, morphine chloride, omeprazole, paroxetine, fluoxetine, Is selected from the group consisting of leucine. In another embodiment, the neoplastic cell represents multiple myeloma (MM), wherein at least one of the chambers is rubicin plus cytarabine + VP-16, daunorubicin plus cytarabine, dirubicin plus cytarabine , Dawsonomy + cytarabine, mitoxantrone + cytarabine + VP-16, ATRA + rubicin, cytarabine + mitoxantrone + ATRA. In another embodiment, the neoplastic cell exhibits chronic lymphocytic leukemia (CLL), wherein at least one of the chamber is selected from the group consisting of cyclophosphamide + doxorubicin + vincristine + prednisolone, cyclophosphamide + doxorubicin + prednisolone, Cyclophosphamide + cyclophosphamide + cyclophosphamide + rituximab, pentostatin + cyclophosphamide + rituximab, fludaravine + cyclophosphamide + oppartum, pentostatin + cyclophosphamide + At least one drug combination selected from the group consisting of daravin + cyclophosphamide + aputujumat, pentostatin + cyclophosphamide + aputujumat. In another embodiment, the neoplastic cell exhibits acute lymphocytic leukemia (ALL), wherein at least one of the chambers is selected from the group consisting of vincristine + daunorubicin + prednisone, vincristine + prednisone + + Cytarabine, methotrexate + cytarabine + hydrocortisone, dexamethasone + vincristine + methotrexate + cytarabine + L-asparaginase + 6-mercaptopurine, cyclophosphamide + doxorubicin + vincristine + dexamethasone, dexamethasone Methotrexate + metoprolol + thioguanine, methotrexate + 6-mercaptopurine, vincristine + daunorubicin + cyclophosphamide + cyclophosphamide + L-asparaginase, vincristine + prednisone, methotrexate + L-Asparaginase + Cyclophosphamide + Prednisona, Ternifoside + Citaravin, Vincristine + Daunorubi + Cyclophosphamide + L-asparaginase + dexamethasone, vincristine + L-asparaginase, vincristine + daunorubicin + cytarabine + L-asparaginase + imatinib + prednisone, mitoxantrone + cytarabine + At least one selected from the group consisting of imatinib, methotrexate + imatinib + 6-mercaptopurina, tenifoside + cytarabine + imatinib, vincristine + daunorubicin + cyclophosphamide + L-asparaginase + dexamethasone + &Lt; / RTI &gt; In another embodiment, the neoplastic cell represents a non-Hodgkin's lymphoma (NHL), wherein at least one of the chambers comprises a cyclophosphamide + doxorubicin + vincristine + prednisone, cyclophosphamide + doxorubicin + Prednisone + Rituximab, Cyclophosphamide + Doxorubicin + Bindesina + Prednisone, Cyclophosphamide + Doxorubicin + Bindesina + Prednisone + Interferon alpha, Cyclophosphamide + Vincristine + Prednisone, Cyclophosphamide + Vincristine + Prednisone + rituximab, mitoxantrone + chlorambucil + prednisolone, mitoxantrone + chlorambucil + prednisolone + rituximab, fludarabine + rituximab, cyclophosphamide + doxorubicin + vindesina + prednisone + Bleomycin, methotrexate + ethofoside + ifosfamide + cytarabine, Methotrexate + vincristine + prednisone, doxorubicin + cyclophosphamide + prednisone +, vincristine + bleomycin + prednisone +, dexamethasone + cytarabine + cisplatin +, fluularabine + cyclophosphamide + mitoxantrone + cyclophosphamide Methotrexate + hydrocortisone + cytarabine + dexamethasone + cyclophosphamide, vendamustine + mitoxantrone, ifosfamide + carboplatin + etoposide + rituximab, etoposide + dexamethasone + dexamethasone + At least one drug combination selected from the group consisting of prednisone + vincristine + cyclophosphamide + doxorubicin + rituximab. In another embodiment, the neoplastic cell exhibits acute myelogenous leukemia (AML), wherein at least one of the chambers is rubicin + cytarabine + VP-16, daunorubicin + cytarabine, dirubicin + (ATRA) + ritonavirin + mitoxantrone + atra (ATRA), daunorubicin + cytarabine + thiobarbituric acid + Guanine, daunorubicin + cytarabine + VP-16, fluarabine + dirubicin + cytarabine + G-CSF, fluarazine + cytarabine + G-CSF, high dose cytarabine + VP- 16 + daunorubicin , Gemtuzumab ozogamycin + rubicin + cytarabine, gemtuzumab ozogamicin + cytarabine, clofarabine + cytarabine, clofarabine + cytarabine + isabicin, amsacrine + cytarabine + VP-16 , Mitoxantrone + VP-16, dirubicin + cytarabine + FLT3 inhibitory stent, cytarabine + FLT3 G-CSF + gaduzumab, cladribine + isrubicin + shitarabine, thalabine + aurora kinase inhibitory stain, dirubicin + cytarabine + panovinostat, fluarabine + dirubicin + cytarabine + Ravin, Decitabine + Valproic acid, Jenasense + Fluidarabine + Citaravin, Jenasense + Daunorubicin + Citaravin, Jenasens + Citarabin, Jenasens + Gentoo Zuma The Ozogami, PSC833 + Daunorubicin At least one drug combination selected from the group consisting of + cytarabine, PSC833 + rubicin + cytarabine, PSC833 + daunorubicin + cytarabine + VP-16, bortezomib + rubidic + cytarabine .

 In another embodiment, a composition for treating chronic lymphocytic leukemia (CLL), comprising fludarabine or a pharmaceutically acceptable salt thereof and sertraline or a pharmaceutically acceptable salt thereof.

 One embodiment includes obtaining a sample of tissue from a serological neoplasm taken from a patient, dividing the tissue sample into at least 35 aliquots, combining the at least 35 fractions with the drug composition, respectively, And measuring apoptosis in at least one cell population within each of the at least 35 fractions. &Lt; RTI ID = 0.0 &gt; [0002] &lt; / RTI &gt;

 Another embodiment is a method of treating a tissue comprising obtaining a sample of tissue obtained from a serological neoplasm taken from a patient and comprising neoplastic cells, dividing the tissue sample into at least 35 aliquots, Wherein the drug composition in each aliquot differs in at least one of the drug composition, concentration, or combination thereof from the drug composition in all other aliquots, and the drug compositions collectively comprise at least one non- Comprising the steps of comprising a toxic drug, culturing the fraction in combination with the drug composition, and analyzing the reactivity of at least one type of neoplastic cell to the drug composition with each cultured aliquot , A method for analyzing the response of a neoplastic tissue to a drug.

 The tissue obtained from the serological neoplasm may be different. For example, the tissue may be selected from the group consisting of peripheral blood, bone marrow, lymph nodes, and spleen. Although it is known to those skilled in the art that the same principles apply to any tissue obtained from the tissues of serological neoplasia containing neoplastic cells, for simplicity, the description herein refers to a blood sample as an example.

 In one embodiment, a method of analyzing cellular response to a drug comprises first obtaining a blood sample taken from the patient; Combining an aliquot of the blood sample with several drug compositions; And analyzing at least one cell population in each of the aliquots for apoptosis. In another embodiment, the blood sample is obtained by a worker and the sample can be sent to another person for analysis. In a preferred embodiment, a method for analyzing a neoplastic blood cell response to a cytotoxic drug, a non-cytotoxic drug, and a combination thereof comprises: a) obtaining a blood sample taken from a patient at a first time point; b) dividing the sample into at least 5, 10, 15, 20, 35, 50, or 100 parts water; c) combining each aliquot with a separate drug composition; d) incubating said fraction with said drug compositions; e) analyzing the response of the at least one neoplastic blood type in the aliquot to the drug composition in the aliquot; And f) completing the method within 48 hours from the time of obtaining the patient blood sample. In another embodiment, the drug composition in combination with each aliquot differs from at least one of the other drugs by at least one of drug congestion, concentration, or a combination thereof.

 In order for a method such as a customized medical examination to function clinically, it is desirable that the method is completed within a short time frame. In particular, the method is completed within a short time frame related to the incubation time of the sample. In one embodiment, the analyzing step is completed within about 120 hours from the time the sample is taken from the patient. In another embodiment, the analyzing step is completed within about 96 hours from the time the sample is taken from the patient. In another embodiment, the analyzing step is completed within about 72 hours from the time the sample is taken from the patient. In another embodiment, the analyzing step is completed within about 48 hours from the time the sample is taken from the patient. In another embodiment, the analysis step is completed within about 24 hours from the time the sample is taken from the patient. However, in other methods, for example, when the sample is frozen or the cells are injected and marketed, the time to complete the assay can be prolonged. In one embodiment, the measuring step is completed within 120 hours of combining the fraction with the drug composition. In another embodiment, the measuring step is completed within 96 hours of combining the aliquot with the drug composition. In another embodiment, the measuring step is completed within 72 hours of combining the aliquot with the drug composition. In another embodiment, the measuring step is completed within 48 hours of combining the aliquot with the drug composition. In another embodiment, the measuring step is completed within 24 hours of combining the aliquot with the drug composition.

Methods for obtaining cell samples from patients are known in the art. In one embodiment, the cell sample is obtained from whole blood. In another embodiment, the cell sample is whole blood. In another embodiment, the cell sample is whole peripheral blood. In another embodiment, the cell sample is obtained from bone marrow. In another embodiment, the cell sample is obtained from a lymph node. In another embodiment, the cell sample is obtained from a spleen. In another embodiment, the cell sample is obtained from any other tissue in combination with serological malignancies. Cell samples can be analyzed as soon as they are obtained, or chemically processed to avoid clotting and analyzed later. In one embodiment, the blood sample is treated with heparin to avoid clotting. In another embodiment, the bone marrow sample is treated with heparin to prevent clotting. In another embodiment, the blood or bone marrow sample is treated with EDTA to prevent clotting. In another embodiment, the blood or bone marrow sample is treated with an anticoagulant including, but not limited to, thrombin to prevent clotting. It is preferred that the sample is used without a purification or separation step so that the cellular environment is more similar to the in vivo environment.

Thousands of drug compositions can be sampled. The methods described herein can assess numerous parameters for customized medical regimens by analyzing numerous combinations of drug compositions in the form of aliquots at various concentrations. In one embodiment, the method comprises administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising about 5,10,15,20,25,30,35,40,45,50,100,200,500 or more aliquots (per drug composition) The range defined by any two of these is analyzed. In another embodiment, the method analyzes 96 or more aliquots. In addition, the number of drug compositions may vary depending on the number of aliquots. In one embodiment, both the number of aliquots and the number of different drug compositions are greater than the range defined by any two of about 5, 10, 15, 20, 25, 30, 35, or 40, Big. In other embodiments, both the number of aliquots and the number of different drug compositions are greater than about 50, respectively. In other embodiments, both the number of aliquots and the number of different drug compositions are greater than 96 each.

The present inventors have unexpectedly discovered that a significant number of non-cytotoxic compounds can induce apoptosis. Although, in some cases, it is known that non-cytotoxic drugs can induce apoptosis in tumor cells, this has been considered a very rare case. The present inventors have found that a significant proportion of non-cytotoxic drugs induce apoptosis in malignant cells from a given serological neoplasm. In addition, the methods described in the present invention unexpectedly demonstrate that certain non-cytotoxic compounds potentiate the ability of cytotoxic compounds to induce apoptosis. Therefore, different types of multiple therapies of multiple drugs may have beneficial therapeutic effects. In one embodiment, the methods described herein analyze cellular responses to drug compositions comprising one or more cytotoxic compounds. In other embodiments, the methods described herein analyze cellular responses to drug compositions comprising one or more non-cytotoxic compounds. In other embodiments, the methods described herein analyze cellular responses to drug compositions comprising one or more cytotoxic compounds and one or more non-cytotoxic compounds. In other embodiments, the methods described herein analyze drug compositions comprising one or more non-cytotoxic compounds of the same or a similar therapeutic category as the drug already administered to the patient. In other embodiments, the methods described herein analyze drug compositions comprising one or more non-cytotoxic compounds in a therapeutic category that is not the same as the drug already administered to the patient. In one embodiment, the drug compositions comprise several compositions comprising the same drug at different concentrations. In other embodiments, the drug composition comprises several different mixtures of drugs. In other embodiments, the drug compositions collectively comprise at least five different drugs.

Potential drug regimens can be optimized for cytotoxic efficiency before administration to the patient. According to the dose response curves generated by the methods described herein for various drug combinations, a lower dose of a highly toxic drug with optimal efficiency can be achieved, indicating synergy between these drugs. Unexpectedly, certain combinations of the two cytotoxic drugs are less effective than the individual drugs, indicating that these cytotoxic drugs behave as cytoprotective agents in certain combinations (i.e., negative co-operative relationships). In one embodiment, the methods described herein utilize optimal conditions to select the drug concentration in the patient. In other embodiments, the methods described herein select drug concentrations for the patient using EC ₉₀ or EC ₅₀ .

In addition to individual drug effects, effective multi-therapy regimens can be identified using detailed analysis of drug interactions, including Combination Index and Dose Reduction Index. The estimates of the accuracy of the two indices are based on a precision algebraic evaluation algorithm based on the median effect described by Chou and Talalay (Chou et al., Adv. Enzyme Regul 1984, 22: 27-55) ). Combination index (CI) is a quantitative measure of the degree of drug interaction in terms of the additive effect, wherein the synergy is represented by CI < 1, the additive effect is represented by CI to I, 1 &lt; / RTI &gt; The dose-reduction index (DRI) is a measure of how much the dose of each drug can be reduced in a synergistic combination, in a given number of effects, relative to the respective drug dose. Recently, the MixLow method (Boik et al., BMC Pharmacol 2008, 8:13; Boik, Stat Med 2008, 27 (7): 1040-61) Effect method (Chou et al., Adv. Enzyme Regul 1984, 22: 27-55). One advantage of the mixrow method is that the nonlinear mixed-effect model used to estimate the parameters of the concentration-response curve is more accurate than the exponential linearization and least-squares analysis used in the median- Can be provided. One of ordinary skill in the art will be able to analyze drug interactions for mixed drug therapies using these calculations and related methods, as described herein. In certain embodiments, the combination of the above medicaments is evaluated for enhancement, synergy or drug abatement. In certain embodiments, combinations identified to demonstrate drug interactions are selected for treatment.

The limited amount of sample that can be extracted from the patient limits the number of drug compositions that can be tested in customized medical examinations. However, due to recent developments, primary cells of patients with serologic malignancies have been propagated through a number of mice to provide a continuous model of mouse cells as a source of patient cells (Pearson et al., Curr Top Microbiol Immunol. 2008, 324: 25-51; Ito et al., Curr Top Microbiol Immunol. 2008, 324: 53-76). This model enables the extracorporeal sample extraction of more drug compositions and especially drug combinations than the recently extracted patient samples. These models are believed to be usable in the method described in the present invention. For example, the sample can be taken from an animal model such as a mouse model. Especially, these. Models enable us to investigate the efficacy of concurrent or adjunctive medications given to patients to alleviate the effects of chemotherapy. These models allow the potential efficacy of approved non-toxic safety drugs to be explored, which can be added to the treatment of individual patients in the future to increase the likelihood of therapeutic efficacy. In addition, the efficacy of any drug combination of drug compositions identified in an in vitro test can be tested in vivo in a live model using human patient cells obtained directly from patient samples or obtained from mouse models.

As a customized medical examination, it is desirable to provide a summary of cellular responses to drug and drug combinations to patients and caregivers. In one embodiment, the method includes creating a report that summarizes the results of the analysis step. In another embodiment, the method comprises providing the patient with the report. In another embodiment, the method comprises providing the report to a party responsible for the medical care of the patient. In another embodiment, the method includes providing a report to a party involved in interpreting the analysis step.

The disclosure of the present invention encompasses certain drug combinations that are useful, for example, in treating AML, ALL, CLL, and NHL, and the use of such drug combinations to treat lymphoproliferative disorders.

 One embodiment provides an apparatus for analyzing the response of neoplastic cells to a potential drug regimen comprising a plurality of chambers and a different drug or drug combination contained in each of the plurality of chambers. In one embodiment, the chambers collectively comprise at least one chamber comprising a plurality of drugs, at least one chamber comprising a cytotoxic drug, and at least a total of 10 different drugs in the collective chambers . In one embodiment, the at least one chamber comprises a non-cytotoxic drug. In one embodiment, the at least one chamber comprises a cytotoxic drug and a non-cytotoxic drug. In one embodiment, at least two chambers contain the same drug at different concentrations.

As will be apparent from the foregoing context, this specification, and the ordinary skill in the art, it is understood that any feature or combination of features described in the invention may be combined with other features, But fall within the scope of the invention. Other advantages and aspects of the present invention will become apparent from the following detailed description.

The present invention relates to the use of screening flats to determine the cytotoxic drug sensitivity properties (cytotoxic drug sensitivity properties) of multiple drug and drug combinations using a sample of cancer patients. The present invention is a cell-based test platform that uses flow cytometry, which is useful for customized medical tests due to its ability to perform rapid data acquisition, analysis, and reporting capabilities from a large number of drug and drug combinations, And an automated evaluation can be performed. Also, the combination of specific drugs described is effective in the treatment of increasing lymphatic diseases.

1 shows flow cytometric analysis of phosphatidylserine expressed on apoptosis cells using the fluorescein labeled annexin V. Fig.
Figure 2 shows the detection of leukemic and normal cells among CD 19 positive cells using a precursor B-ALL adult event and quantitative flow cytometry showing BCR / ABL gene rearrangement [t (9; 22) positive].
Figure 3 shows the norm for in vitro evaluation of peripheral blood (PB) or bone marrow (BM) in samples of patients with chronic lymphocytic leukemia (CLL).
Figure 4 shows the in vitro response to several drugs currently approved for CLL therapy in nine different patients.
Figure 5 shows the number of drug compositions desired to optimize customized medical examinations for individual patients.
Figure 6 depicts a number of non-invasive methods for inducing apoptosis with similar potency to cytotoxic drugs approved for CLL therapy in malignant CLL samples (e.g., fluaravarin, chlormbucil, and mitoxantrone) - Cytotoxic drugs (eg, paroxetine, fluoxetine, sertaline, guanabenz, and astemizole).
FIG. 7 shows a dose-response curve for paroxetine in a whole blood sample from a CLL patient and compares the apoptosis effect of paroxetine on leukemic, T, and NK cells.
Figure 8 shows the difference in the mechanical difference between the induction of apoptosis by sertraline and the three drugs currently used in CLL treatment (e.g., fluaravarin, chlorambucil, and mitoxantrone) in a CLL patient whole blood sample .
Figure 9 shows the differential efficacy of compounds in the same pharmacological grade (eg, SSRIs) as paroxetine in inducing apoptosis in CLL samples.
Figure 10 shows the frequency of hits as the number of patient samples in which a non-cytotoxic drug removes leukemic CLL cells with the same potency as an approved cytotoxic drug, and most non-cytotoxic drugs It seems to be effective for very few patients.
Figure 11 shows the enhanced potency of approved drug cytotoxic chlorambucil by the non-cytotoxic drug sertraline.
Figure 12 shows the percentage of annexin V-positive cells induced by the cytotoxic drugs vincristine, mitoxantrone, and cyclophosphamide (used for CLL treatment) and the non-cytotoxic drugs omeprazole and acyclovir Lt; / RTI &gt; positive cells that are often prescribed to treat side effects caused by therapy.
Figure 13 shows a 96-well plate design for customized medical examinations of CLL patients.
Figure 14 shows a 96-well plate design for customized medical examinations of CLL patients.
Figure 15 shows a 96-well plate design for customized medical examinations of CLL patients.
Figure 16 shows a 96-well plate design for customized medical examination of multiple myeloma patients.
Figure 17 shows a 96-well plate design for customized medical examination of multiple myeloma patients.
Figure 18 shows a 96-well plate design for customized medical examination of multiple myeloma patients.
Figure 19 shows a 96-well plate design for customized medical examination of multiple myeloma patients.
Figure 20 shows a 96-well plate design for customized medical examination of multiple myeloma patients.
Figure 21 shows a 96-well plate design for customized medical examination of multiple myeloma patients.
Figure 22 shows a 96-well plate design for customized medical examinations of acute lymphocytic leukemia (ALL) patients, including cytotoxic and non-cytotoxic drugs administered as a treatment standard for PETHEMA. MTX: methotrexate; 6MP: 6-mercaptopurine; ARA-C: cytarabine; DNR: Daunorubicin; ADRIA: Adriamycin; M: mitoxantrone; VP-16: ethofoside; VM-26: tennaphoside; CF: cyclophosphamide; IFOS: ifosfamide; V: Vincristine; VIND: Vindeshin; L-ASA: asparaginase; IMAT: Imatinib; R: rituximab; P: prednisone; HC: hydrocortisone; DXM: dexamethasone; FoIi: Luco borin; Mesna: Messina; Om: omeprazole; O: ondansetron; Allop: allopurinol; GCSF: The Phil Grass team.
Figure 23 shows a 96-well plate design for customized medical examinations of patients with myelodysplastic syndromes, including cytotoxic and non-cytotoxic drugs administered as a treatment standard for PETHEMA.
Figure 24 depicts a 96-well plate design for customized medical examination of patients with acute myelogenous leukemia (other than M3), including cytotoxic and non-cytotoxic drugs administered as a treatment standard for PETHEMA. Dauno: Daunorubicin; Ida: Is Ruby Shin; ARA-C: cytarabine; Mitox: mitoxantrone; VP16: ethofoside; Fluda: fludaravin; GCSF: Peel Grass Team; Ondans: ondansetron; Cotri: Co-tree pastor; AcF: folic acid; Alop: allopurinol; Om: omeprazole; Carbop: Carboplatin; Dauno lipo: liposomal daunorubicin (Daunosomes); AMSA: Amsacrine; GO: Gentoo Gomez Ozogamisina.
Figure 25 shows a 96-well plate design for customized medical examination of patients with acute myelogenous leukemia M3 (Promyelocytic), including cytotoxic and non-cytotoxic drugs administered as a treatment standard for PETHEMA . ATRA (all-trans retinoic acid): tretinoin; Ida: Is Ruby Shin; Mitox: mitoxantrone; ARA-C: cytarabine; 6-MP: 6-mercaptopurine; MTX: methotrexate; Ondans: ondansetron; Alop: allopurinol; Om: omeprazole; Dexa: dexamethasone; VP-16: ethofoside; Fluda: fludaravin; Carbop: Carboplatin; Dauno lipo: liposomal daunorubicin; Dauno: Daunorubicin; Cotri: Co-tree pastor; FAc: Folic acid.
Figure 26 shows the effect of sertraline on inhibition of cell proliferation in TOM-I and MOLT-4 cell lines.
Figure 27 shows the effect of sertraline on the induction of apoptosis at 24 hours in TOM-I and MOLT-4 cell lines.
Figure 28 shows the effect of sertraline on induction of active caspase-3 at 24 hours in TOM-I and MOLT-4 cell lines.
Figure 29 is a graph showing the effect of the most intrinsic and susceptible multireflexion of CLL samples using individual drugs (e.g., rituxamip, fludarabine, mitoxantrone, and cyclophosphamide (maposphamide)) and combinations of these individual drugs Indicates in vitro efficacy.
Figure 30 shows the results of the same experiment as in Figure 29 with a 5-point dose response curve characterizing the in vitro efficacy of fluaravarin, cyclophosphamide (maposphamide), and combinations thereof.
Figure 31 shows the results of the same experiment as in Figure 30 with a 5-point dose response curve characterizing the in vitro efficacy of fluaravarin, cyclophosphamide (maposphamide), mitoxantrone, and combinations thereof.
Figure 32 shows the results of the same experiment as in Figure 31 with a 5-point dose response curve characterizing the in vitro efficacy of fluaravarin, cyclophosphamide (maposphamide), rituximab, and combinations thereof.
Figure 33 shows the effect of treating fludaravin and mapposmide alone and in combination at five different concentrations as clinical criteria for two patients P2.0144 (left) and P2.0149 (right).
Figure 34 shows the synergism between fluaravarin and mappospha mide (cyclophosphamide), found in CLL patient P2.0149 of Figure 33, and the Talaray method (Chou et al., Eur J Biochem 1981, 115 (l) 207-16; Chou et al., Adv. Enzyme Regul 1984, 22: 27-55).
Figure 35 shows the incubation times (drug exposure time (0.5, 4, and 8 hours) and total incubation time (24 hours) for the efficacy of fluaravarin and sertraline to induce apoptosis in malignant cells in CLL samples Or 48 hours).
Figure 36 shows a matrix of two drug combinations.
Figure 37 shows a matrix of three drug combinations.
Figure 38 shows a matrix of four drug combinations.
Figure 39 shows the 3-color multiplexing of peripheral blood leukocytes using a cell tractor dye.
Figure 40 shows fluorescent dye dyes used in multiplex wells in CLL samples to differentiate malignant cells and detect Annoptin V apoptosis.

 The present invention provides compositions, systems and methods for evaluating the in vitro apoptotic potency of multiple drug combinations using a screening platform. In particular, the present invention provides a method for performing cell-based screening that includes both automatic sample preparation and automated evaluation by flow cytometry, which is suitable for rapid data acquisition, analysis and reporting of results. Using flow cytometry, individual cell death can be assessed and its single cell assay results in reduced incubation time for in vitro assays, thereby providing faster turnover rates for cytotoxic profiling. The cell-based screening platform can also be used to complete all screening and validation analyzes within 24-72 hours of sample extraction. These timetables will allow the serologic neoplasm to be reported to the physician prior to the start of treatment after a diagnostic analysis has been performed. In conclusion, the method described in this invention can be used in customized medicine and allows identification of possible new prescriptions for approved drugs.

For a better understanding of the present invention, certain terms are first defined. Additional definitions are set forth in the detailed description.

The terms &quot; EC ₅₀ &quot; and &quot; EC ₉₀ &quot;, as used herein, refer to the drug concentrations required to elicit 50% and 90% of maximal apoptosis, respectively.

The term &quot; in vitro &quot; as used herein refers to a primary human patient cell in a test tube that has been immediately extracted, frozen, or frozen to preserve its condition. In one embodiment, it is thawed for in vitro evaluation of drug efficacy.

As used herein, the term &quot; in vitro therapeutic index &quot; refers to the ratio between neoplastic and healthy cell death.

The term &quot; Exvitech &quot; as used herein refers to an integrated platform incorporating automated sample preparation, an EPS system for automated input into flow cytometry analyzers, and automated biometric analysis.

 The term &quot; serologic neoplasm &quot; as used herein also refers to a group of diseases, referred to as &quot; serological malignancies &quot;, defined by the World Health Organization classification (Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri S, Stein H, Thiele J, Vardiman JW (Eds): WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues, International Agency for Research in Cancer (IARC), Lyon.

 The term personalized tumor response test / assay (ITRT) as used herein refers to a method of demonstrating the effect of chemotherapy on the whole tumor cell line taken from a cancer patient.

 The term &quot; multi-therapy &quot; as used herein refers to the treatment of a patient with multiple drugs.

 The term &quot; non-toxic &quot; compound or drug used in the present invention refers to a compound or drug that is not approved as cytotoxic, chemotherapeutic, or anti-neoplastic agent by the regulatory authorities.

 As used herein, the term &quot; fraction &quot; may be in a separate container or well, or even in a suitable sample, whether the sample is continuous or separated by a gas or an immiscible liquid (e.g., oil) Refers to a sample or fraction thereof that may be formed in a tube or other medium where differences in drug content, drug congestion or drug concentration may be maintained.

 The term &quot; drug composition &quot; as used herein refers to a single drug, and various concentrations thereof, or combinations of media and various concentrations thereof, administered to a patient for administration or for treatment, for analysis.

 The term &quot; pharmaceutically acceptable salts &quot; as used herein refers to preparations of the compounds that do not cause severe irritation to the administered organism and do not interfere with the biological activity and properties of the compound. Pharmaceutical salts can be obtained through routine experimentation.

 As used herein, the term &quot; well &quot; or &quot; chamber &quot; refers to any structure with a capacity to float sufficient sample to perform the method described herein. Those skilled in the art know that the &quot; well &quot; or &quot; chamber &quot; is, for example, a concave portion on a plate, a point on a glass slide, or a portion of a microfluidic device caused by surface tension.

 Reference will now be made to preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same or similar reference numerals are used in the drawings and the description to refer to the same or like parts. It should be noted that the drawings are in simplified form and are not to scale. Although the disclosure of the present invention refers to specific illustrated embodiments, these embodiments are presented by way of illustration and not limitation of the invention. The intent of the following detailed description is to be construed as including all alterations, alterations, and equivalents, which may be included within the spirit and scope of the present invention, as defined by the appended claims, even if the exemplary embodiment is discussed. The disclosed methods can be used in conjunction with various medical methods commonly used in the art.

 The disclosed methods have several advantages over the prior art discussed in the present invention. One is that these methods can analyze cellular responses to a number of variables, including many drug compositions and different incubation times. Another advantage is the rate at which the method analyzes the cellular response to the drug. Another advantage is the ability to analyze whole blood, thus replicating the patient's in vivo environment more precisely. In addition, the methods can produce dose response curves for a number of drug and drug compositions. Once combined, these methods offer the advantage of developing a multimodal regimen to treat the patient. In certain embodiments, the methods facilitate the development of a multireflex regimen for treating patients with serological disorders.

 The disclosed methods can be modified with the use of various types of equipment, including one or more sample preparation automated machines (robots) and one or more flow cytometry analyzers to analyze cellular response to drug compositions. Flow cytometry enables single cell analysis at a rate that far exceeds any other single cell analysis technique in the art. This makes it possible to analyze a statistically significant number of cells faster than using other alternative techniques. In one embodiment, flow cytometry is used to analyze cellular response to drug compositions. In one embodiment, the assay is completed within 96 hours from the time the sample is obtained. In another embodiment, the analysis is completed within 72 hours from the time the sample is obtained. In another embodiment, the analysis is completed within 48 hours from the time the sample is obtained. In another embodiment, the analysis is completed within 24 hours from the time the sample is obtained. An example of a flow cytometer useful in the methods described herein is provided in U.S. Patent No. 7,459,126, the contents of which are hereby incorporated by reference in their entirety for all purposes, including the object of describing flow cytometry analyzers as a whole and without limitation.

 The sample manufacturing robot and the flow cell analyzer may be integrated with each other, or the sample manufacturing robot and the flow cell analyzers may not be integrated with each other. In one embodiment, a flow cytometer can be used without a sample preparation robot. In certain embodiments, a CYAN (TM) cell analyzer (Beckman Coulter, Fullerton, Calif.) Is used without a sample preparation robot. There are various types of sample making robots and liquid handling machines known in the art. In one embodiment, the flow cytometer is used with any suitable sample preparation robot or liquid handling machine known in the art. In another embodiment, the CYAN (TM) cell analyzer (Beckman Coulter, Fullerton, Calif.) Integrates with a small liquid handling unit called EPS and automatically transfers the sample to a cell analyzer. In certain embodiments, the EPS is a Tecan 360 liquid handling machine (Tecan, Mannedorf, Switzerland). In another embodiment, the EPS comprises a syringe pump and an interface switching valve that allows the contents of each well to be drawn through a fixed tip, delivered into a holding loop, and injected into the cell analyzer. In other embodiments, sample preparation and compound plate dispensing can be accomplished using a BIOMEK 3000 liquid dispenser (Beckman Coulter, Fullerton, Calif.). Any number of well plates may be used, and particularly useful well plates are 96-well plates. Various other plate sizes are also contemplated, including those with 24, 48, 384, 1536, 3456, or 9600 wells. Sample preparation units may be packaged in a flow cabinet that allows the compounds and samples to remain sterile while being treated. Once the analysis equipment organization is complete, load the plate into the sample analysis system.

 A CYAN (TM) cell analyzer (Beckman Coulter, Fullerton, Calif.) Is a three-optical-nine detector instrument and it is well known in the art how to fully utilize the multi-lasers of such modern flow cytometry analyzers and, consequently, have. In one embodiment, a single laser flow cytometer is used in the analysis step. In another embodiment, a multi-laser flow cytometer is used in the analysis step. It has become possible to develop and optimize a vast array of fluorochromes and conjugation chemistries to allow a variety of ligands such as immunoglobulins and small molecules to be conjugated to the fluorescent dye. Lasers with emission lines in the ultraviolet to red region of the light spectrum can excite such fluorescent pigments. In conclusion, a number of spectroscopic reagents can be used to label cells to study fluorescence-based instrumentation, such as flow cytometry. Such reagents are known in the art. In one embodiment, one or more fluorescent pigments are used during the analysis step. In certain embodiments, one or more dyes are used to analyze cellular responses to a drug composition.

 There are several methods known in the art that minimize inadvertent sample mixing in the tube prior to analysis. One such method known to minimize inadvertent sample mixing during in vitro analysis is a positive displacement pump that allows all tubes to be washed between wells to prevent cell rollovers. Another useful method to minimize careless sample mixing is the endpoint assay. End point analysis is advantageous because compound carryover is not a problem. In one embodiment, the method comprises an endpoint assay.

 There are several methods known in the art to collect and store data obtained using flow cytometry analyzers. In one embodiment, the software installed in the EPS records time information for injection and incubation times for each well. When operating the screening method, two acquisition files can be collected. In one embodiment, one file is located in the cell analyzer software, which contains actual data for each cell analyzed by the instrument. In another embodiment, the second file is a timing file, which is present in the EPS software or in the cell analyzer software, and contains actual data for each cell analyzed by the device. Each of these files may be named according to a barcode scanned from a well plate, e.g., a 96-well plate, at the beginning of the assay run.

 The user can load all of the files from both devices into an analysis software program such as EPS Analyzer. These programs divide the data obtained from the cell analyzer into several groups and give each cell a well number of compounds mixed with the cells. Another use of the program is to allow each individual group to be analyzed separately, including checking individual groups based on fluorescence readings. Analytical markers included in the assay device are also evaluated. In one embodiment, for screening and confirmatory analysis, annexin V FITC, a marker of apoptosis associated with fluorophores, is used to distinguish viable cells entering the apoptosis pathway.

 After the analysis is complete, upload the files to the database. In one embodiment, the database is ACTIVITYBASE (TM) from IDBS (Guildford, UK). Uploading the file to the database quickly evaluates the data to determine the active compound in each patient sample. As the data accumulates, the bioinformatics means are dried and developed to facilitate data interpretation. As an example, pharmacological criteria, such as EC50, EC90, maximum apoptosis, etc., obtained from acquired data can be compared across many patient samples and correlated with immune expression trait results and genetic information. Considering the large amount of data acquired on each analysis screen, a flexible database management system is critical to the screening process.

 This system allows the determination of in vitro therapeutic indices by measuring the ability of the drug composition to induce apoptosis. Figures 1 and 2 show the ability to detect apoptotic cells using flow cytometry and the ability to distinguish between normal and tumor phenotypes. In one embodiment, the methods use flow cytometry to distinguish between normal and tumor phenotypes. In another embodiment, the method distinguishes between normal and tumor phenotypes using flow cytometry and monoclonal antibodies. In another embodiment, the method detects apoptotic cells using flow cytometry. In certain embodiments, the method detects phosphatidylserine expression on apoptosis cells using Annexin V conjugated with fluorescein isothiocyanate (FITC). Using multiple combinations of the appropriate combinations of monoclonal antibodies known in the art with a multivariate analysis strategy allows the distinction of residual normal and leukemic cells present in the sample from patients with serological disorders. In one embodiment, the method enables the distinction between malignant and normal cells in a blood or bone marrow sample. In another embodiment, the distinction between malignant and normal cells in the blood or bone marrow is provided by the EuroFlow Consortium (Cytometry A. 2008 Sep; 73 (9): 834-46; van Dongen et al., 14th EHA Congress, Berlin, DE 4 June 2009 (planned to be published in Leukemia 2010 (published)).

 An in vitro screening method for the drug composition is schematically illustrated in FIG. In Fig. 3, the sample is inhibited from clotting by heparin, immunoprecipitated, and counted. The sample is then diluted to obtain a leukemic cell concentration of about 4,000 cells / μL. 45 [mu] l of the cell suspension is added to 96-well plates containing the pharmaceutical preparation at five different concentrations. After the drug and drug combination is incubated with the sample for about 48 hours, the red blood cells are dissolved and washed to concentrate the leukocytes containing the malignant cells. This facilitates the screening process by dramatically reducing the volume and number of cells that need to be evaluated by flow cytometry analyzers. Fluorescently labeled antibodies are added to distinguish malignant cells from healthy cells, and fluorescence labeled annexin V is added to measure the level of apoptosis in each cell population, such as in malignant cells. The following screening is performed, the activity of each drug composition is determined, and the results are analyzed and reported.

 In one embodiment, the method comprises dividing the sample into aliquots and dispensing the aliquots into a well plate. These well plates contain individual drugs or drug combinations at various concentrations. In one embodiment, the well plates have individual drugs or combinations at various concentrations prior to introducing the cell sample. In other embodiments, the cell samples are introduced into the wells before individual drugs or combinations are introduced at various concentrations. In another embodiment, compounds of the formula (I) may be present in the range of about 20, 30, 50, 75, 100, 200, 300, 500, 700, 1000 or 2000 compounds, An enormous library of the included compounds can be used. In certain embodiments, the aliquots contain diseased cells per well as detectable water. In one embodiment, the aliquots contain at least about 500 diseases or neoplastic cells per well. In other embodiments, the aliquots contain more than about 5,000 diseases or neoplastic cells per well. In other embodiments, the aliquots contain at least about 10,000 diseases or neoplastic cells per well. In other embodiments, the aliquots contain more than about 20,000 diseases or neoplastic cells per well. In other embodiments, the aliquots contain more than about 40,000 diseases or neoplastic cells per well. Sample testing can be done in duplicate. In one embodiment, at least two aliquots can be tested in parallel to determine the immunophenotype. In addition, a natural level of apoptosis not associated with drug therapy can be identified, including control wells without any drug (not shown). In one embodiment, the method identifies a natural level of apoptosis in the sample using control wells.

 The duration of incubation with different aliquots of different drug compositions varies. In one embodiment, the period is up to about 24 hours. In another embodiment, the period is up to about 48 hours. In other embodiments, the period is up to about 72 hours. In another embodiment, the period is up to about 96 hours. In other embodiments, the period is up to about 120 hours. After incubation for a specific period of time, a sample aliquot exposed to the drug composition can be treated with a buffer to dissolve the red blood cell population and concentrate the white blood cell population. In one embodiment, a population of erythrocytes can be lysed using buffers known in the art. Each of the following wells is cultured with a reagent for detecting apoptosis by flow cytometry. In one embodiment, the reagent is Annexin V.

 Using flow cytometry, it is possible to assess the effect of each drug on each cell type and to quantify the level of selective apoptosis induced by each drug. A new test can be started with additional samples or aliquots to evaluate the following results and, if necessary, to ascertain in more detail the most appropriate results, such as the ten highest drug compositions and concentrations already identified. After the analysis is performed on a patient sample, a suitable drug or drug composition that can selectively induce apoptosis in neoplastic cells such as leukemic cells can be selected. In one embodiment, about 5-20 drug compositions were identified and re-tested with fresh samples. In certain embodiments, the five highest drug compositions are identified and retested with fresh samples. In another particular embodiment, the 10 highest drug compositions are identified and retested with a fresh sample. In another particular embodiment, the 20 highest drug compositions are identified and retested with a fresh sample.

 A number of drugs currently approved for chronic lymphocytic leukemia (CLL) were analyzed in various patients using the methods provided herein. For example, the efficacy of an individually approved cytotoxic drug in inducing apoptosis in malignant cells in an ex vivo patient sample is provided in FIG. Figure 4 high individual-to-person variability in drug response, highlighting the potential of the method described in this invention as a customized medical examination.

 In one embodiment, the method identifies a drug composition that elicits greater than 90% Apoptosis in a patient sample. In another embodiment, the method identifies a drug composition that induces at least 75% apoptosis in a patient sample. In another embodiment, the method identifies a drug composition that induces at least 50% apoptosis in a patient sample.

 Figure 4 shows that the methods described in the present invention can also detect drug compositions that do not generally induce apoptosis in patient samples. Inability to induce apoptosis may be a genetic predisposition of the patient to drug resistance or may be the result of inherent resistance of the neoplasm to the drug. For any reason, the ability to predict the inability of the drug composition to induce apoptosis is desirable. In one embodiment, the method identifies a drug composition that induces less than 90% of apoptosis in a patient sample. In another embodiment, the method identifies a drug composition that induces less than 75% of apoptosis in a patient sample. In another embodiment, the method identifies a drug composition that induces less than 50% of apoptosis in a patient sample. In another embodiment, the method identifies a drug composition that induces less than 30% of apoptosis in a patient sample.

 It is an advantageous feature of the method of the present invention that a whole sample, such as a whole peripheral blood or bone marrow sample, is recently obtained, treated with heparin for anti-coagulation, and diluted if necessary. In one embodiment, the method of the invention uses a blood sample. In another embodiment, the method of the invention uses a whole blood sample. In another embodiment, the method of the invention uses a whole peripheral blood sample. In another embodiment, the method of the invention uses a bone marrow sample. In one embodiment, the method of the invention uses a sample taken from an animal model. In one embodiment, the method of the invention uses a sample taken from a mouse model. The entire sample is advantageous because conventional in vitro tests contain tumor cells and only isolate mononuclear fractions that discard the corresponding polymorphonuclear leukocytes, red blood cells, proteins and other plasma factors through washing. This severely alters the biological context in which the drug's effects are evaluated. On the other hand, the method of the present invention can retain proteins such as albumin, which normally binds about 90% to 98% of each drug as well as erythrocytes in plasma.

 Thus, the drug concentration used in the assay described herein can be considered closer to the actual drug concentration present in the patient &apos; s plasma. Using the entire sample is also important because it makes it easier to observe the effects of antibodies such as camphor or rituximab in inducing apoptosis in tumor cells. Other measures such as the percentage of cell depletion may also be important than the percentage of apoptosis. Although both methods measure apoptosis, cell depletion counts cells that are no longer alive for a drug-free control aliquot. Direct apoptosis detection counts cells undergoing apoptosis during measurement. The difference is the number of cells entering the necrotic state after apoptosis and the number of cells no longer detectable by the flow cytometer. Depending on the measurement time, these two methods report different results. For example, at shorter detection times (e.g., 24 hours), cell depletion and apoptosis are similar. However, at longer detection times (e. G., 48 to 72 hours), these measures split as the number of cells initially experiencing apoptosis and no longer being detected increases. In order to induce rituximab apoptosis, in vitro analysis requires a complement found in the mononuclear fraction that is usually removed. In conclusion, the method of the present invention allows the original cellular microenvironmental conditions to be maintained at a greater level in the sample being analyzed. In one embodiment, the method of the present invention substantially maintains the original cell microenvironment.

 The automated flow cytometry platform described herein is the first platform to screen numerous drug composition variables in in vitro patient serological samples. These platforms enable exploration of multiple drug combinations for the induction of apoptosis in individual patients. Since serologists generally only use drugs and drug combinations that have previously been formally recognized (e.g., as proven through clinical trials) in the treatment protocol, And evaluation of drug and drug combinations in treatment modalities. These treatment modalities may include recognized norms in certain countries. Such treatment norms may also include older acceptance norms, although they are no longer the preferred treatment norms. Newer experimental protocols (eg, those that are still at the clinical trial stage) may also be included, including approved drugs and new drug compositions of drugs on clinical phase II or III. The methods and devices described in the present invention can also be used to evaluate combinations of drugs, including those on approved drugs and on Clinical &lt; RTI ID = 0.0 &gt; II &lt; / RTI &

 The previously used ITRT in vitro tests to induce tailored patient treatment were limited to individual drugs or very few drug combinations. The advantage of evaluating multiple drug combinations, for example, using the ExviTech platform, is shown in FIG. In FIG. 29, in the case of CLL patient samples, individual CLL drugs were not effective (on the left), indicating ineffective treatment. However, these same drugs produced three combinations, which were very effective at removing all leukemic cells (right), indicating potential as a sensitive therapy. Thus, evaluating individual drugs or drug combinations used in current treatment regimens could have been counterproductive. Figure 29 shows information that can be of great importance for effective treatment of serologic neoplasms (a tolerance rule that predicts a lack of clinical response (center) and a very sensitive rule (right) that predicts favorable clinical responses).

 In certain embodiments, the custom medical exam described herein evaluates five different concentrations of each drug or drug combination. This allows the determination of a minimum dose-response curve that provides more accurate pharmacological efficacy determinations than single dose data. It also facilitates qualitative management by analyzing whether five points fit the sigmoid capacity-response curve. The same data described above in Figure 29 for the CLL sample is shown in the 5-point dose-response curve in Figures 30-32.

 In an in vitro evaluation of a drug combination, it is important to determine whether there is positive or negative co-operability between the combined drugs, referred to as synergism. Such in vitro co-operation can illustrate in vivo cooperation in the patient. If there is a positive synergy between drugs, the potency is likely to increase compared to the higher therapeutic index, toxicity. If there is a highly toxic, cytotoxic drug, increasing their therapeutic index is therapeutically important. Therefore, there are several ways to quantify drug synergism, and the most commonly used methods are the Chu and Talaray methods (Chou et al., Adv. Enzyme Regul 1984, 22: 27-55). Figure 33 shows a synergistic combination of fluaravarin and mappospamide (metabolites and active ingredients of cyclophosphamide) in two CLL patient samples, wherein the cointegration index (CI) was calculated using the program Calcusyn (Chou et al., Adv Enzyme Regul 1984, 22: 27-55) to characterize potential synergism with the combination. Figure 34 shows a more elaborate calculation of the synergy found in patient P2.0149 from Figure 33 using the weight and Talaray method.

 The efficacy of drug and drug combinations can also be influenced by their kinetics. Figure 35 shows the different kinetic behavior of the approved cytotoxic drug fluaravarin and non-cytotoxic antidepressant sertraline in CLL samples. Serratralin (right panel) removes all malignant cells within 25 hours (right upper panel), while fluarabine requires 48 hours (left lower panel). However, both drugs require only 30 minutes of incubation time to induce maximum apoptosis. This indicates that malignant cells are programmed to undergo apoptosis in short-term cultures, even though the apoptosis measured by anesin V requires 24 or 48 hours to be fully detected. In one embodiment, the drug compositions are incubated for a period of time of about 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, or any two of the above- do. In other embodiments, apoptosis is measured at about 24 hours, 48 hours, or 72 hours after incubation, or at any of the two defined ranges of time values.

 As the methodology described in the present invention has been demonstrated, it is possible to evaluate hundreds to thousands of individual wells containing serological samples mixed with drugs showing different composition and concentration. The limits of drug compositions are governed not by the throughput of the platform but by the amount of serological samples obtained from the patient and the cellularity. For each drug composition, up to about 10,000 or more drug compositions per sample obtained, or more than 20,000 per sample in a sample having more than a typical amount of sample, due to the small amount used, such as about 20,000 cells per well It is possible to evaluate the drug composition. The number of such combinations is sufficient to evaluate alternative multireflex drug compositions that can be administered to individual patients. In one embodiment, such assays are performed with a minimal amount of about 500 neoplastic cells per well. In other embodiments, these assays are performed with a minimal amount of about 1,000 neoplastic cells per well. In other embodiments, these assays are performed with a minimal amount of about 20,000 neoplastic cells per well. In other embodiments, these assays are performed with a minimal amount of about 50,000 neoplastic cells per well. The number of malignant cells per patient sample can vary from practically zero to one billion, and therefore their relative proportional value to the total number of cells can also vary.

 Figure 5 shows the number of potential drug compositions that can be explored to identify optimal multimetric therapies for individual patients. Hypothetically, up to 15 drugs approved for a particular prescription are shown in the first column on the left hand side in FIG. There are many drugs available for serological neoplasms, and some newer drugs are expected to be approved within the next few years. In addition, many non-cytotoxic drugs are given to patients with serologic neoplasms (eg, concurrent medications) to alleviate the effects of cytotoxic therapy, usually ranging from 5 to 10 drugs per patient. These drugs range from gastrointestinal protectants to anti-vomiting anti-vaginal agents, and anti-infective antibiotics and antiviral agents. The fifteen drugs selected in Fig. 5 are marked as a large number of approved cytotoxic drugs that can be considered in a given prescription and, therefore, can be considered representative of a particular clinical practice. The selection of the 15 drugs in Figure 5 is for illustrative purposes only and should not be construed as a limitation of the invention for any reason. In Figure 5, a combination of up to four different drugs (the second column) has been presented as a representative average number, even if there are norms to combine 5 and 6 drugs. The design of the well plates described herein demonstrates the use of up to 22 different drugs in a single 96 well plate. Also, plates having different numbers of wells can be used to analyze different numbers of drugs. In one embodiment, about five drugs are selected and analyzed. In other embodiments, about 10 drugs are selected and analyzed. In other embodiments, about 40 drugs are selected and analyzed. In another embodiment, about 20 drugs are selected and analyzed.

 As illustrated in Figure 5, the number of different combinations for 15 drugs in the second column is 1940 and 1470 for 14 drugs. These results may also be used to inform treatment decisions that they are preferably performed at a concentration of 5 per drug or drug combination (third column). Furthermore, evaluation of at least two incubation times enables evaluation of kinetic parameters (fourth column). However, assay performance at five doses and / or at least one incubation time should not be construed as a limitation.

 Despite all of the variables discussed in the preceding paragraph (e.g., number of different drugs, multiple measurements, various drug concentrations, and various incubation times), automated platforms capable of evaluating up to about 10,000 or 20,000 drug compositions, It will be able to take care of all of the hypothetical scenarios described as the missing parts. Due to the therapeutic space, it is possible to study the gray areas in FIG. 5 in the current method. Drug compositions that can be developed by up to 30 to 35 current methods are painted gray. The remainder of the above table represents the new space of the drug composition that can be developed by the ExviTech platform. Currently available passive platforms are limited to their potential use as customized medical examinations because only about 35 individual conditions can be assessed. The automation of drug effects in in vitro serological samples is suitable and innovative for customized medical applications. In one embodiment, the method analyzes only 1,000 drug compositions. In another embodiment, the method analyzes about 10,000 drug compositions. In another embodiment, the method analyzes less than 20,000 drug compositions. In certain embodiments, the methods described herein enable the analysis of up to about 20,000 drug compositions, which are responsible for one, two, three, or four drug combinations of up to 15 drugs. In certain embodiments, the incubation time also varies. For example, as shown in Figure 5 (4th column), inclusion of more than one incubation time increases the number of combinations to be tested. In certain embodiments, the use of ExviTech allows measurement of the non-obscure areas in Figure 5, which represents most of the drug compositions required for customized treatment of the patient.

 The ExviTech platform can also be used to screen thousands of drugs, especially about 1,000 approved drugs per patient sample, to search for drugs that selectively induce apoptosis in malignant cells. Surprisingly, a number of approved non-cytotoxic drugs have been shown to induce apoptosis in malignant cells with the same potency as the approved cytotoxic drugs for each formulation. Figure 6 shows how the five non-cytotoxic drugs, unapproved for serological malignancies, remove CLL malignant cells with similar efficacy (right) with the three approved cytotoxic CLL drugs (on the left). Figure 7 shows the dose response of the antidepressant paroxetine, one such drug, demonstrating that the drug induces apoptosis in malignant B cells more favorably than healthy T and NK cells. Figure 8 shows how sertraline, one of these non-cytotoxic drugs, clears malignant CLL cells faster than the approved cytotoxic CLL drug (24 vs. 48 hours) (left). Three of the five most effective non-cytotoxic drugs are the antidepressant drugs paroxetine, fluoxetine, and sertraline (drugs belonging to the same pharmacological group). Figure 9 shows how only three out of six serotonin reuptake inhibitors are efficient at inducing apoptosis in malignant CLL cells. This indicates that these effects are not necessarily related to the pharmaceutical grade of the drug and that the in vitro customized medical tests presented in the present invention can be used to elucidate such activity.

 The unexpected discovery that a number of safe non-cytotoxic approved drugs may be efficacious against tumor cells has facilitated a wider assessment. First, several such drugs were effective in any given sample, discarding non-selective effects. Figure 10 is drawn from screening of 2,000 drugs in 23 CLL samples showing how the efficacy of these approved non-cytotoxic drugs varies considerably from patient to patient. Drugs are defined as effective if they kill more than 80% of malignant cells, a criterion similar to the most effective cytotoxic drugs. Only 3 drugs were effective in over 80% of patients, while 229 drugs were effective in less than 20% of patient samples. This indicates that although non-cytotoxic drugs may be effective against in vitro malignant cells, patient-to-patient variability is very high. Nevertheless, in almost all CLL patient samples, 5-10 non-cytotoxic drugs have been found to be effective in ex vivo malignant cells. Although the predictability of this effect is not known in vivo, as other factors such as pharmacokinetics and formulation work strongly, the effect of 5-10 such non-cytotoxic drugs administered to the patient presents a significant therapeutic benefit .

 Some of these ex vivo effective non-cytotoxic drugs are drugs used to alleviate the effects of cytotoxic drugs administered to patients with serological malignancies (e.g., concurrent medications). Figure 12 shows an example of a CLL sample showing significant efficacy against ex vivo malignant cells, similar to the efficacy of proton pump undeclared omeprazole and antiviral acyclovir in the efficacy of cytotoxic drugs. Table 4 lists some of these co-administered drugs

<Concurrent Administration> drug Symptom Aluminum oxide hydrate Antacid Laurajempam Antidepressant Amikacin Antibiotics (aminoglucoside) Meropenem Antibiotics (Beta-lactam) Cephalam Antibiotics (cephalosporin) Vancomycin Antibiotics (glycopeptides) Teicoplanin Antibiotics (glycopeptides) Ondansetron Jet Dexamethasone Anti-inflammatory, immunosuppressive, glucocorticoid Amphotericin B (liposomal)  Antifungal Carposfugin  Antifungal Itraconazole  Antifungal Fluconazole  Antifungal Boriconeazole  Antifungal Trimethoprim & Bacteriostats  Sulfamethoxazole G-CSF Granulocyte colony stimulating factor Lanitidine Histamine H2-receptor antagonist Rasburikaze Hyperuricemia treatment Paracetamol Non-steroidal anti-inflammatory Metamizole Non-steroidal anti-inflammatory Morphine chloride Hypoactive analgesic Omeprazole Decomposition of proton pump Paroxetine Antidepressant Fluoxetine Antidepressant Sertraline Antidepressant

 Some of these non-cytotoxic approved drugs are therapeutically effective, enhancing the effect of cytotoxic drugs (e.g., as a chemosensitising agent). An example is shown in FIG. 11, where a low concentration of antidepressant sertraline enhances the efficacy of the low-level cytotoxic drug chlorambucil.

 The method of the present invention was intended to analyze a number of variables, and 96-well plates were devised to explore potential parameters in multimetrically therapy. Other plates with larger or smaller numbers of wells may also be used. In one embodiment, 1536 well plates are used. In other embodiments, 384 well plates are used. In other embodiments, 96 well plates are used.

 Figures 13-25 and Examples 9-14 illustrate the use of a 96 well plate format to analyze patient samples for the following indications: chronic lymphocytic leukemia, acute lymphocytic leukemia, multiple myeloma, myelodysplastic syndrome, acute myelogenous leukemia Subacute leukemia (not M3), and acute myelogenous leukemia M3. According to the plate design for each prescription, the Spanish program for serologic malignancy treatment for signs (Programa para el Tratamiento de Hemopatias Malignas (PETHEMA) Use a drug.

 In one embodiment, the method analyzes drugs selected from approved norms of the clinic. In certain embodiments, the method analyzes a drug selected from the PETHEMA treatment standard. In accordance with the well planning, a monotherapy-prescribed drug is used under the PETHEMA treatment standard and a combination of monotherapy drugs is used. Also, according to the scheme, the prescribed medicines are used to alleviate the side effects of the PETHEMA treatment standard, and a combination of these drugs is also used.

 In one embodiment, the method analyzes cytotoxic drugs, including approved drugs and drugs not yet approved for clinical trials. In another embodiment, the method analyzes a combination of cytotoxic drugs. In another embodiment, the method analyzes drugs prescribed for treating side effects of cytotoxic drugs. In another embodiment, the method analyzes a combination of drugs prescribed to treat a side effect of a cytotoxic drug. The wells also use cytotoxic drugs and a combination of drugs prescribed to treat the side effects of cytotoxic drugs. In one embodiment, the method analyzes a combination of cytotoxic drugs and a combination of drugs prescribed to treat the side effects of cytotoxic drugs. In another embodiment, the method analyzes any and all non-cytotoxic drugs prescribed in any and all indications that are approved or in clinical trials. In another embodiment, the method analyzes a combination of non-cytotoxic drugs. For example, combinations of cytotoxic drugs and non-cytotoxic drugs may be used, depending on the plate approach. In one embodiment, the method analyzes a combination of cytotoxic drugs and non-cytotoxic drugs.

 Treatment for serologic neoplasms is determined by a limited number of treatment modalities agreed by the serologist. These norms define a multireflex regimen for both cytotoxicity and additional combination drugs, including the dose and time of administration of each drug. These norms are dependent on variables such as age, well-being and disease status of each patient. Norms may also vary from country to country, but are well observed within a country. There are still considerable variations within these norms in terms of dosages and different drug compositions requiring hundreds of conditions to be studied. In one embodiment, clinically proven reagents are used to assess cellular apoptosis. In other embodiments, clinically proven reagents are used in combination with antibodies to identify subtypes of tumor cells. In other embodiments, the reagents used to identify subtypes of tumor cells have recently been described in the Euroflow standard (van Dongen et al., Euroflow Antibody Panel for Normalized, Reactive and Malignant Leukocyte Normalized, n-Dimensional Flow- 14th EHA Congress, Berlin, DE 4 June 2009: scheduled to be published in Leukemia 2010 (published)). In other embodiments, the drug compositions may be selected from the norms for serological therapy used in a particular country. In other embodiments, the drug compositions are selected from outdated norms for serological therapy used in a particular country. In other embodiments, the drug compositions are selected from experimental norms, defined as new combinations of approved drugs used in a particular country. In other embodiments, the drug compositions are selected from those comprising drugs that are evaluated in clinical trials for serological treatments.

 The effects of each drug used in the treatment regimen are desirable to be studied individually. However, such studies should not be construed as limitations. Monotherapy drugs should be studied individually using the methods of the present invention, as well as drugs that are conventionally administered only as a combination. For example, individual screening of drugs that are administered only as a combination will enable the individual effects of these drugs to be determined.

 In one embodiment, monotherapy drugs are analyzed individually. In other embodiments, drugs that are generally administered only as a combination are analyzed separately. Non-cytotoxic drugs commonly used in conjunction with cytotoxic drugs should also be studied (e.g., as in FIG. 12). This includes antibiotics, aerosols (antiseptics), antacids, antiviral agents and the like. In one embodiment, the method analyzes the ability of omeprazole to induce apoptosis in a patient sample. In another embodiment, the method analyzes the ability of acyclovir to induce apoptosis in a patient sample.

 Indeed, it has been demonstrated using the methods of the present invention that non-cytotoxic drugs such as paroxetine and sertraline can potentiate the efficacy of modulating the effects of cytotoxic drugs such as fludarabine and chlorambucil, respectively , As shown in Fig. 11). As shown in Figure 6, some of these non-cytotoxic drugs administered alone lead to in vitro apoptosis in malignant cells, similar to the efficacy of an approved cytotoxic drug. In one embodiment, the method uses a non-cytotoxic drug to induce apoptosis in a patient sample. In another embodiment, the method uses a combination of non-cytotoxic drugs to induce apoptosis in a patient sample. In another embodiment, the method uses a combination of cytotoxic and non-cytotoxic drugs to induce apoptosis in a patient sample.

 Unexpectedly, certain non-cytotoxic drugs have eliminated malignant cells, without harm to healthy cells, indicating that such drugs selectively attack malignant cells (e.g., as shown in FIG. 12). Such unexpected results have far-reaching implications for the treatment of serological neoplasms. In one embodiment, the method selectively induces apoptosis in neoplastic cells using non-cytotoxic drugs. In one embodiment, the in vitro therapeutic index is greater than about 1. In other embodiments, the in vitro therapeutic index is greater than about 5. In other embodiments, the in vitro therapeutic index is greater than about 10. In certain embodiments, the methods described herein enable differentiation between leukemic and normal cells in tissues associated with serological neoplasms, such as blood, bone marrow, lymph nodes, or spleen samples.

 In addition, the ability of a non-cytotoxic drug to induce apoptosis varies within the pharmacological rating of the drug (e.g., as shown in Figure 9) as well as between the pharmacological ratings of the drug. In one embodiment, the method analyzes drugs selected from the same pharmacological rating as the drug administered to the patient for treatment of a particular condition or to alleviate side effects of treatment of a particular condition. In certain embodiments, the method analyzes selective serotonin reabsorbtion degradations. In addition, the method of the present invention is not limited to the analysis of only cytotoxic drugs or only non-cytotoxic drugs. Indeed, there are cases where a combination of non-cytotoxic drug and cytotoxic drug may have a greater ability to induce apoptosis in a patient sample relative to the ability of the cytotoxic drug alone (e.g., 11).

 The system of the present invention is capable of fully analyzing the combination of two classes of drugs, such as cytotoxic and non-cytotoxic drugs generally administered together. In one embodiment, the non-cytotoxic drug prescribed is administered to the patient to which the cytotoxic drug is administered. For example, the methods of the invention can be used to analyze patient samples treated with cytotoxic drug fludaravines and non-cytotoxic selective serotonin reuptake inhibitors. In certain embodiments, the method is used to analyze a patient sample treated with the cytotoxic drug fluaravine and the non-cytotoxic drug paroxetine. In the case of serologic neoplasia, the patient drug regimen may include multiple drug combinations. In one embodiment, the drugs prescribed for serological symptoms are analyzed in various combinations. For example, each patient receives an average of 8 to 10 drugs. In one embodiment, five or more drug compositions are analyzed. A preferred scheme of the plate for some of the major symptoms is shown in the following examples.

 The current strategy of norm-based treatment for serologic neoplasms is the result of a drug development stagnation. Because of this downturn, serologists became familiar with certain drugs and developed a rational assessment of the best combination of each drug. In one embodiment, the methods described in the present invention demonstrate current scientific expectations for drug compositions. However, two factors are dramatically changing the current strategy of norm-based therapy. First, as shown in FIG. 4, each patient responds differently to chemotherapy, and the fact is that recently customized medicine has been at the forefront of medical research. Molecular biology techniques and the revolution brought on by the human genome detoxification are focused on genomic analysis of patient samples with serological neoplasms. However, genome studies of 10 to 15 years have stratified patients at risk, but did not tailor treatment to individual patients. These actual results have led to the desire to use customized medical examinations to combine optimal therapy with individual patients. However, current norms are estimated to study less than 1-5% of the available treatment space that the platform of the present invention can examine (as shown in FIG. 5). Next, there are a number of new drugs recently approved for serologic neoplasms, and there are also some late stage clinical candidates. In conclusion, these diseases are rapidly moving from scenarios of the same old drugs prescribed over the years to scenarios using many new drugs approved within the next few years. In one embodiment, the methods of the invention are used to evaluate old drugs, new drugs, late stage clinical candidates, or combinations thereof.

 The methods of the present invention are useful for identifying drug choices on a personalized patient basis and for identifying trends in treatment modalities that would be useful for selecting drugs for patients having similar symptoms and responses to current drug regimens. All patients will have these compounds in the blood and bone marrow at selected concentrations to remove malignant cells. One advantage of the multifunctional customized medical exam described herein is the ability to study many different drug compositions that simultaneously administer 8 to 10 drugs. In one embodiment, multiple drugs are co-administered to a patient. In other embodiments, multiple drugs are administered serially to the patient. Many of the drugs provided in the present invention have not been evaluated for combination administration. As shown in the following examples, a clear and dramatic effect on the induction of apoptosis is observed for these drugs in combination.

 Another advantage is the ability to determine the optimal drug composition on a personalized basis. As shown in FIG. 10, the patient's response to certain drug compositions varies widely. In fact, only three drugs induced apoptosis in over 80% of neoplastic cells over 80% of the 23 patient samples. In contrast, 229 different drugs induced apoptosis in over 80% of neoplastic cells for less than 20% (1-4) patient samples. This indicates that most non-cytotoxic drugs are effective in very few patients and that there is greater individual-to-person variation than for cytotoxic drugs. However, serologic neoplasms patients are usually administered 5-10 non-cytotoxic concurrent drugs to relieve the effects of cytotoxic drugs. Thus, among these concurrent drugs, the additional effect of selecting a subgroup that exhibits significant efficacy in inducing in vitro apoptosis of malignant cells, as in Figure 12, may be significant.

 In addition to identifying the potentially most efficacious drug for individual patients, these results can also group patients into small groups and, for these subgroups, including cytotoxic and non-cytotoxic drugs, Can be presented. In one embodiment, the medication treatment standard is selected on an individual patient basis. In other embodiments, the drug treatment standard is selected based on its efficacy in 1-4 patient samples. In other embodiments, the drug treatment standard is selected based on its efficacy in 5-9 patient samples. In other embodiments, the drug treatment standard is selected based on its efficacy in 10 to 14 patient samples. In other embodiments, the drug treatment standard is selected based on its efficacy in 15-19 patient samples. In other embodiments, the drug treatment standard is selected based on its efficacy in more than 20 patient samples. The method of the present invention gives more options than currently available treatment norms.

 One advantage of customized medical examinations is the ability to optimize certain drug regimens on an individual basis. In multi-therapy regimens where several different drugs are administered to a patient in combination, the pharmacokinetics and general dose response curves of the individual drugs are not typical. Using the methods of the present invention, neoplastic and optimal doses for normal cells can be observed by recognizing optimal in a dose response curve for a particular patient.

 A variety of drug and drug combinations can be used in the methods and apparatus described herein. For example, drug combinations comprising cytotoxic drugs can be used. Also, drug combinations comprising non-cytotoxic drugs can be used. Furthermore, drug combinations of cytotoxic and non-cytotoxic drugs can be used

 Examples of cytotoxic compounds that may be used alone or in combination with other compounds include fludarabine (designated "1"), chlorambucil (designated "2"), mitoxantrone (designated "3" (Designated "4"), mitoxantrone (designated "5"), cyclophosphamide (designated "6"), adriamycin (designated "7"), and doxorubicin Designation).

 Specific examples of non-cytotoxic compounds which may be used alone or in combination with other compounds include, but are not limited to, 5-azacytidine (designated as "1"), alemtuzumab (designated as "2"), aminopterin (Designated "4"), Amasacrine (designated "5"), CAT-8015 (designated "6"), bevacizumab (designated "7"), ARR Y520 (Designated "9"), AS1413 ("10"), ATRA ("11"), AZD 6244 ("12"), AZDl 152 (18 &quot;), &lt; / RTI &gt; bovine mothyl (- (24), chlorophenol (21), CEP-701 (22), chlorambucil (23), chlorodeoxyadenosine (24) ), Cyclophosphamide ("28"), cyclosporin ("29"), cladribine (" ), Desaturin (&quot; 34 &quot;), deglycosylated (&quot; 32 &quot;), cytarabine (&quot; 30 &quot;), cytosine arabinoside Lysine-A chain-conjugated anti-CD 19 / anti-CD22 immunotoxins ("35"), dexamethasone ("36"), doxorubicin ("37" (&Quot; 39 &quot;), eftatuzumab (&quot; 40 &quot;), (47), gemcitabine (&quot; 48 &quot;), gemtuzumab-ozogamicin (&quot; 49 &quot;), homoharring (50), hydrocortisone (51), hydroxycarbamide (52), dirubicin (53), ifosfamide (54), imatinib Isoindole I 131 monoclonal antibody BC8 (&quot; 57 &quot;), isosfamide (&quot; 58 &quot;), iso (60), L-asparaginase (61), lanalidomide (62), lestaurinib (63) (&Quot; 64 &quot;), methylprednisone (&quot; 69 &quot;), midosutaurin (&quot; (&Quot; 70 &quot;), mitoxantrone (&quot; 71 &quot;), nelarabine (&quot; 72 &quot;), neurotinib ("77"), pentostatin ("78"), pyruvicin ("79"), PKC412 ("80"), prednisolone (83), Rituximab (84), Ribavirin (85), Sapathi Tabin (86), Dinashi Clip (87) , Sorafenip ("88"), Sorapenip ("89"), STA-9090 ("90"), Tacrolimus ("91" ("93"), tenofoside ("94"), terameprocool ("95"), thalidomide ("96"), thioguanine ("99"), topotecan ("100"), threosulfan ("101"), trloxathiabine ("102"), vinblastine ("105"), vinorelbine ("106"), borreloxin ("107"), borinostat (" .

 Specific examples of non-cytotoxic compounds that can be used alone or in combination with other compounds include, but are not limited to, aluminum oxide hydrate ("111"), lorazepam ("112"), amikacin (" 114), amphotericin B (liposomes), amphotericin B (&quot; Liposomes &quot;), (120), carposofugine (121), itraconazole (122), fluconazole (123), voriconazole (124), trimethoprim (125) (126), G-CSF (127), ranitidine (128), rasbicase (129), paracetamol (130), metamizole (131) ("132"), omeprazole ("133"), paroxetine ("134"), fluoxetine ("135") and sertraline ("136"

 In addition, in most countries, certain drug combinations represent the preferred or standard cytotoxic therapy for AML, ALL, CLL, and NHL therapy. These existing therapies may be assigned numerical designators and may be used in further combinations with other additional drugs in the following combinations.

 Using the above numerical designators in the form #. #, An example of a combination of two compounds containing at least one cytotoxic compound is described below, which may or may not further include combinations of other compounds:

One. 2, 1. 3, 1. 4, 1. 5, 1. 6, 1. 7, 1. 8, 1. 9, 1. 10, 1. 11, 1. 12, 1. 13, 1. 14, 1. 15, 1. 16, 1. 17, 1. 18, 1. 19, 1. 20, 1. 21, 1. 22, 1. 23, 1. 24, 1. 25, 1. 26, 1. 27, 1. 28; One. 29, 1. 30, 1. 31, 1. 32, 1. 33, 1. 34, 1. 35, 1. 36, 1. 37, 1. 38, 1. 39, 1. 40, 1. 41, 1. 42, 1. 43, 1. 44, 1. 45, 1. 46, 1. 47, 1. 48, 1. 49, 1. 50, 1. 51, 1. 52, 1. 53, 1. 54, 1. 55, 1. 56, 1. 57, 1. 58, 1. 59, 1. 60, 1. 61, 1. 62, 1. 63, 1. 64, 1. 65, 1. 66, 1. 67, 1. 68, 1. 69, 1. 70, 1. 71, 1. 72, 1. 73, 1. 74, 1. 75, 1. 76, 1. 77, 1. 78, 1. 79, 1. 80, 1. 81, 1. 82, 1. 83, 1. 84, 1. 85, 1. 86, 1. 87, 1. 88, 1. 89, 1. 90, 1. 91, 1. 92, 1. 93, 1. 94, 1. 95, 1. 96, 1. 97, 1. 98, 1. 99, 1. 100, 1. 101, 1. 102, 1. 103, 1. 104, 1. 105, 1. 106, 1. 107, 1. 108, 1. 109, 1. 110, 1. 111, 1. 112, 1. 113, 1. 114, 1. 115, 1. 116, 1. 117, 1. 118, 1. 119, 1. 120, 1. 121, 1. 122, 1. 123, 1. 124, 1. 125, 1. 126, 1. 127, 1. 128, 1. 129, 1. 130, 1. 131, 1. 132, 1. 133, 1. 134, 1. 135, 1. 136; 2. 3, 2. 4, 2. 5, 2. 6, 2. 7, 2. 8, 2. 9, 2. 10, 2. 11, 2. 12, 2. 13, 2. 14, 2. 15, 2. 16, 2. 17, 2. 18, 2. 19, 2. 20, 2. 21, 2. 22, 2. 23, 2. 24, 2. 25, 2. 26, 2. 27, 2. 28; 2. 29, 2. 30, 2. 31, 2. 32, 2. 33, 2. 34, 2. 35, 2. 36, 2. 37, 2. 38, 2. 39, 2. 40, 2. 41, 2. 42, 2. 43, 2. 44, 2. 45, 2. 46, 2. 47, 2. 48, 2. 49, 2. 50, 2. 51, 2. 52, 2. 53, 2. 54, 2. 55, 2. 56, 2. 57, 2. 58, 2. 59, 2. 60, 2. 61, 2. 62, 2. 63, 2. 64, 2. 65, 2. 66, 2. 67, 2. 68, 2. 69, 2. 70, 2. 71, 2. 72, 2. 73, 2. 74, 2. 75, 2. 76, 2. 77, 2. 78, 2. 79, 2. 80, 2. 81, 2. 82, 2. 83, 2. 84, 2. 85, 2. 86, 2. 87, 2. 88, 2. 89, 2. 90, 2. 91, 2. 92, 2. 93, 2. 94, 2. 95, 2. 96, 2. 97, 2. 98, 2. 99, 2. 100, 2. 101, 2. 102, 2. 103, 2. 104, 2. 105, 2. 106, 2. 107, 2. 108, 2. 109, 2. 110, 2. 111, 2. 112, 2. 113, 2. 114, 2. 115, 2. 116, 2. 117, 2. 118, 2. 119, 2. 120, 2. 121, 2. 122, 2. 123, 2. 124, 2. 125, 2. 126, 2. 127, 2. 128, 2. 129, 2. 130, 2. 131, 2. 132, 2. 133, 2. 134, 2. 135, 2. 136; 3. 4, 3. 5, 3. 6, 3. 7, 3. 8, 3. 9, 3. 10, 3. 11, 3. 12, 3. 13, 3. 14, 3. 15, 3. 16, 3. 17, 3. 18, 3. 19, 3. 20, 3. 21, 3. 22, 3. 23, 3. 24, 3. 25, 3. 26, 3. 27, 3. 28; 3. 29, 3. 30, 3. 31, 3. 32, 3. 33, 3. 34, 3. 35, 3. 36, 3. 37, 3. 38, 3. 39, 3. 40, 3. 41, 3. 42, 3. 43, 3. 44, 3. 45, 3. 46, 3. 47, 3. 48, 3. 49, 3. 50, 3. 51, 3. 52, 3. 53, 3. 54, 3. 55, 3. 56, 3. 57, 3. 58, 3. 59, 3. 60, 3. 61, 3. 62, 3. 63, 3. 64, 3. 65, 3. 66, 3. 67, 3. 68, 3. 69, 3. 70, 3. 71, 3. 72, 3. 73, 3. 74, 3. 75, 3. 76, 3. 77, 3. 78, 3. 79, 3. 80, 3. 81, 3. 82, 3. 83, 3. 84, 3. 85, 3. 86, 3. 87, 3. 88, 3. 89, 3. 90, 3. 91, 3. 92, 3. 93, 3. 94, 3. 95, 3. 96, 3. 97, 3. 98, 3. 99, 3. 100, 3. 101, 3. 102, 3. 103, 3. 104, 3. 105, 3. 106, 3. 107, 3. 108, 3. 109, 3. 110, 3. 111, 3. 112, 3. 113, 3. 114, 3. 115, 3. 116, 3. 117, 3. 118, 3. 119, 3. 120, 3. 121, 3. 122, 3. 123, 3. 124, 3. 125, 3. 126, 3. 127, 3. 128, 3. 129, 3. 130, 3. 131, 3. 132, 3. 133, 3. 134, 3. 135, 3. 136; 4. 5, 4. Six, four. 7, 4. 8, 4. 9, 4. 10, 4. 11, 4. 12, 4. 13, 4. 14, 4. 15, 4. 16, 4. 17, 4. 18, 4. 19, 4. 20, 4. 21, 4. 22, 4. 23, 4. 24, 4. 25, 4. 26, 4. 27, 4. 28; 4. 29, 4. 30, 4. 31, 4. 32, 4. 33, 4. 34, 4. 35, 4. 36, 4. 37, 4. 38, 4. 39, 4. 40, 4. 41, 4. 42, 4. 43, 4. 44, 4. 45, 4. 46, 4. 47, 4. 48, 4. 49, 4. 50, 4. 51, 4. 52, 4. 53, 4. 54, 4. 55, 4. 56, 4. 57, 4. 58, 4. 59, 4. 60, 4. 61, 4. 62, 4. 63, 4. 64, 4. 65, 4. 66, 4. 67, 4. 68, 4. 69, 4. 70, 4. 71, 4. 72, 4. 73, 4. 74, 4. 75, 4. 76, 4. 77, 4. 78, 4. 79, 4. 80, 4. 81, 4. 82, 4. 83, 4. 84, 4. 85, 4. 86, 4. 87, 4. 88, 4. 89, 4. 90, 4. 91, 4. 92, 4. 93, 4. 94, 4. 95, 4. 96, 4. 97, 4. 98, 4. 99, 4. 100, 4. 101, 4. 102, 4. 103, 4. 104, 4. 105, 4. 106, 4. 107, 4. 108, 4. 109, 4. 110, 4. 111, 4. 112, 4. 113, 4. 114, 4. 115, 4. 116, 4. 117, 4. 118, 4. 119, 4. 120, 4. 121, 4. 122, 4. 123, 4. 124, 4. 125, 4. 126, 4. 127, 4. 128, 4. 129, 4. 130, 4. 131, 4. 132, 4. 133, 4. 134, 4. 135, 4. 136; 5. Six, five. 7, 5. 8, 5. 9, 5. 10, 5. 11, 5. 12, 5. 13, 5. 14, 5. 15, 5. 16, 5. 17, 5. 18, 5. 19, 5. 20, 5. 21, 5. 22, 5. 23, 5. 24, 5. 25, 5. 26, 5. 27, 5. 28; 5. 29, 5. 30, 5. 31, 5. 32, 5. 33, 5. 34, 5. 35, 5. 36, 5. 37, 5. 38, 5. 39, 5. 40, 5. 41, 5. 42, 5. 43, 5. 44, 5. 45, 5. 46, 5. 47, 5. 48, 5. 49, 5. 50, 5. 51, 5. 52, 5. 53, 5. 54, 5. 55, 5. 56, 5. 57, 5. 58, 5. 59, 5. 60, 5. 61, 5. 62, 5. 63, 5. 64, 5. 65, 5. 66, 5. 67, 5. 68, 5. 69, 5. 70, 5. 71, 5. 72, 5. 73, 5. 74, 5. 75, 5. 76, 5. 77, 5. 78, 5. 79, 5. 80, 5. 81, 5. 82, 5. 83, 5. 84, 5. 85, 5. 86, 5. 87, 5. 88, 5. 89, 5. 90, 5. 91, 5. 92, 5. 93, 5. 94, 5. 95, 5. 96, 5. 97, 5. 98, 5. 99, 5. 100, 5. 101, 5. 102, 5. 103, 5. 104, 5. 105, 5. 106, 5. 107, 5. 108, 5. 109, 5. 110, 5. 111, 5. 112, 5. 113, 5. 114, 5. 115, 5. 116, 5. 117, 5. 118, 5. 119, 5. 120, 5. 121, 5. 122, 5. 123, 5. 124, 5. 125, 5. 126, 5. 127, 5. 128, 5. 129, 5. 130, 5. 131, 5. 132, 5. 133, 5. 134, 5. 135, 5. 136; 6. 7, 6. 8, 6. 9, 6. 10, 6. 11, 6. 12, 6. 13, 6. 14, 6. 15, 6. 16, 6. 17, 6. 18, 6. 19, 6. 20, 6. 21, 6. 22, 6. 23, 6. 24, 6. 25, 6. 26, 6. 27, 6. 28; 6. 29, 6. 30, 6. 31, 6. 32, 6. 33, 6. 34, 6. 35, 6. 36, 6. 37, 6. 38, 6. 39, 6. 40, 6. 41, 6. 42, 6. 43, 6. 44, 6. 45, 6. 46, 6. 47, 6. 48, 6. 49, 6. 50, 6. 51, 6. 52, 6. 53, 6. 54, 6. 55, 6. 56, 6. 57, 6. 58, 6. 59, 6. 60, 6. 61, 6. 62, 6. 63, 6. 64, 6. 65, 6. 66, 6. 67, 6. 68, 6. 69, 6. 70, 6. 71, 6. 72, 6. 73, 6. 74, 6. 75, 6. 76, 6. 77, 6. 78, 6. 79, 6. 80, 6. 81, 6. 82, 6. 83, 6. 84, 6. 85, 6. 86, 6. 87, 6. 88, 6. 89, 6. 90, 6. 91, 6. 92, 6. 93, 6. 94, 6. 95, 6. 96, 6. 97, 6. 98, 6. 99, 6. 100, 6. 101, 6. 102, 6. 103, 6. 104, 6. 105, 6. 106, 6. 107, 6. 108, 6. 109, 6. 110, 6. 111, 6. 112, 6. 113, 6. 114, 6. 115, 6. 116, 6. 117, 6. 118, 6. 119, 6. 120, 6. 121, 6. 122, 6. 123, 6. 124, 6. 125, 6. 126, 6. 127, 6. 128, 6. 129, 6. 130, 6. 131, 6. 132, 6. 133, 6. 134, 6. 135, 6. 136; 7. 8, 7. 9, 7. 10, 7. 11, 7. 12, 7. 13, 7. 14, 7. 15, 7. 16, 7. 17, 7. 18, 7. 19, 7. 20, 7. 21, 7. 22, 7. 23, 7. 24, 7. 25, 7. 26, 7. 27, 7. 28; 7. 29, 7. 30, 7. 31, 7. 32, 7. 33, 7. 34, 7. 35, 7. 36, 7. 37, 7. 38, 7. 39, 7. 40, 7. 41, 7. 42, 7. 43, 7. 44, 7. 45, 7. 46, 7. 47, 7. 48, 7. 49, 7. 50, 7. 51, 7. 52, 7. 53, 7. 54, 7. 55, 7. 56, 7. 57, 7. 58, 7. 59, 7. 60, 7. 61, 7. 62, 7. 63, 7. 64, 7. 65, 7. 66, 7. 67, 7. 68, 7. 69, 7. 70, 7. 71, 7. 72, 7. 73, 7. 74, 7. 75, 7. 76, 7. 77, 7. 78, 7. 79, 7. 80, 7. 81, 7. 82, 7. 83, 7. 84, 7. 85, 7. 86, 7. 87, 7. 88, 7. 89, 7. 90, 7. 91, 7. 92, 7. 93, 7. 94, 7. 95, 7. 96, 7. 97, 7. 98, 7. 99, 7. 100, 7. 101, 7. 102, 7. 103, 7. 104, 7. 105, 7. 106, 7. 107, 7. 108, 7. 109, 7. 110, 7. 111, 7. 112, 7. 113, 7. 114, 7. 115, 7. 116, 7. 117, 7. 118, 7. 119, 7. 120, 7. 121, 7. 122, 7. 123, 7. 124, 7. 125, 7. 126, 7. 127, 7. 128, 7. 129, 7. 130, 7. 131, 7. 132, 7. 133, 7. 134, 7. 135, 7. 136; 8. 9, 8. 10, 8. 11, 8. 12, 8. 13, 8. 14, 8. 15, 8. 16, 8. 17, 8. 18, 8. 19, 8. 20, 8. 21, 8. 22, 8. 23, 8. 24, 8. 25, 8. 26, 8. 27, 8. 28; 8. 29, 8. 30, 8. 31, 8. 32, 8. 33, 8. 34, 8. 35, 8. 36, 8. 37, 8. 38, 8. 39, 8. 40, 8. 41, 8. 42, 8. 43, 8. 44, 8. 45, 8. 46, 8. 47, 8. 48, 8. 49, 8. 50, 8. 51, 8. 52, 8. 53, 8. 54, 8. 55, 8. 56, 8. 57, 8. 58, 8. 59, 8. 60, 8. 61, 8. 62, 8. 63, 8. 64, 8. 65, 8. 66, 8. 67, 8. 68, 8. 69, 8. 70, 8. 71, 8. 72, 8. 73, 8. 74, 8. 75, 8. 76, 8. 77, 8. 78, 8. 79, 8. 80, 8. 81, 8. 82, 8. 83, 8. 84, 8. 85, 8. 86, 8. 87, 8. 88, 8. 89, 8. 90, 8. 91, 8. 92, 8. 93, 8. 94, 8. 95, 8. 96, 8. 97, 8. 98, 8. 99, 8. 100, 8. 101, 8. 102, 8. 103, 8. 104, 8. 105, 8. 106, 8. 107, 8. 108, 8. 109, 8. 110, 8. 111, 8. 112, 8. 113, 8. 114, 8. 115, 8. 116, 8. 117, 8. 118, 8. 119, 8. 120, 8. 121, 8. 122, 8. 123, 8. 124, 8. 125, 8. 126, 8. 127, 8. 128, 8. 129, 8. 130, 8. 131, 8. 132, 8. 133, 8. 134, 8. 135, 8. 136; 9. 10, 9. 11, 9. 12, 9. 13, 9. 14, 9. 15, 9. 16, 9. 17, 9. 18, 9. 19, 9. 20, 9. 21, 9. 22, 9. 23, 9. 24, 9. 25, 9. 26, 9. 27, 9. 28; 9. 29, 9. 30, 9. 31, 9. 32, 9. 33, 9. 34, 9. 35, 9. 36, 9. 37, 9. 38, 9. 39, 9. 40, 9. 41, 9. 42, 9. 43, 9. 44, 9. 45, 9. 46, 9. 47, 9. 48, 9. 49, 9. 50, 9. 51, 9. 52, 9. 53, 9. 54, 9. 55, 9. 56, 9. 57, 9. 58, 9. 59, 9. 60, 9. 61, 9. 62, 9. 63, 9. 64, 9. 65, 9. 66, 9. 67, 9. 68, 9. 69, 9. 70, 9. 71, 9. 72, 9. 73, 9. 74, 9. 75, 9. 76, 9. 77, 9. 78, 9. 79, 9. 80, 9. 81, 9. 82, 9. 83, 9. 84, 9. 85, 9. 86, 9. 87, 9. 88, 9. 89, 9. 90, 9. 91, 9. 92, 9. 93, 9. 94, 9. 95, 9. 96, 9. 97, 9. 98, 9. 99, 9. 100, 9. 101, 9. 102, 9. 103, 9. 104, 9. 105, 9. 106, 9. 107, 9. 108, 9. 109, 9. 110, 9. 111, 9. 112, 9. 113, 9. 114, 9. 115, 9. 116, 9. 117, 9. 118, 9. 119, 9. 120, 9. 121, 9. 122, 9. 123, 9. 124, 9. 125, 9. 126, 9. 127, 9. 128, 9. 129, 9. 130, 9. 131, 9. 132, 9. 133, 9. 134, 9. 135, 9. 136; 10. 11, 10. 12, 10. 13, 10. 14, 10. 15, 10. 16, 10. 17, 10. 18, 10. 19, 10. 20, 10. 21, 10. 22, 10. 23, 10. 24, 10. 25, 10. 26, 10. 27, 10. 28; 10. 29, 10. 30, 10. 31, 10. 32, 10. 33, 10. 34, 10. 35, 10. 36, 10. 37, 10. 38, 10. 39, 10. 40, 10. 41, 10. 42, 10. 43, 10. 44, 10. 45, 10. 46, 10. 47, 10. 48, 10. 49, 10. 50, 10. 51, 10. 52, 10. 53, 10. 54, 10. 55, 10. 56, 10. 57, 10. 58, 10. 59, 10. 60, 10. 61, 10. 62, 10. 63, 10. 64, 10. 65, 10. 66, 10. 67, 10. 68, 10. 69, 10. 70, 10. 71, 10. 72, 10. 73, 10. 74, 10. 75, 10. 76, 10. 77, 10. 78, 10. 79, 10. 80, 10. 81, 10. 82, 10. 83, 10. 84, 10. 85, 10. 86, 10. 87, 10. 88, 10. 89, 10. 90, 10. 91, 10. 92, 10. 93, 10. 94, 10. 95, 10. 96, 10. 97, 10. 98, 10. 99, 10. 100, 10. 101, 10. 102, 10. 103, 10. 104, 10. 105, 10. 106, 10. 107, 10. 108, 10. 109, 10. 110, 10. 111, 10. 112, 10. 113, 10. 114, 10. 115, 10. 116, 10. 117, 10. 118, 10. 119, 10. 120, 10. 121, 10. 122, 10. 123, 10. 124, 10. 125, 10. 126, 10. 127, 10. 128, 10. 129, 10. 130, 10. 131, 10. 132, 10. 133, 10. 134, 10. 135, 10. 136; 11. 12, 11. 13, 11. 14, 11. 15, 11. 16, 11. 17, 11. 18, 11. 19, 11. 20, 11. 21, 11. 22, 11. 23, 11. 24, 11. 25, 11. 26, 11. 27, 11. 28; 11. 29, 11. 30, 11. 31, 11. 32, 11. 33, 11. 34, 11. 35, 11. 36, 11. 37, 11. 38, 11. 39, 11. 40, 11. 41, 11. 42, 11. 43, 11. 44, 11. 45, 11. 46, 11. 47, 11. 48, 11. 49, 11. 50, 11. 51, 11. 52, 11. 53, 11. 54, 11. 55, 11. 56, 11. 57, 11. 58, 11. 59, 11. 60, 11. 61, 11. 62, 11. 63, 11. 64, 11. 65, 11. 66, 11. 67, 11. 68, 11. 69, 11. 70, 11. 71, 11. 72, 11. 73, 11. 74, 11. 75, 11. 76, 11. 77, 11. 78, 11. 79, 11. 80, 11. 81, 11. 82, 11. 83, 11. 84, 11. 85, 11. 86, 11. 87, 11. 88, 11. 89, 11. 90, 11. 91, 11. 92, 11. 93, 11. 94, 11. 95, 11. 96, 11. 97, 11. 98, 11. 99, 11. 100, 11. 101, 11. 102, 11. 103, 11. 104, 11. 105, 11. 106, 11. 107, 11. 108, 11. 109, 11. 110, 11. 111, 11. 112, 11. 113, 11. 114, 11. 115, 11. 116, 11. 117, 11. 118, 11. 119, 11. 120, 11. 121, 11. 122, 11. 123, 11. 124, 11. 125, 11. 126, 11. 127, 11. 128, 11. 129, 11. 130, 11. 131, 11. 132, 11. 133, 11. 134, 11. 135, 11. 136; 12. 13, 12. 14, 12. 15, 12. 16, 12. 17, 12. 18, 12. 19, 12. 20, 12. 21, 12. 22, 12. 23, 12. 24, 12. 25, 12. 26, 12. 27, 12. 28; 12. 29, 12. 30, 12. 31, 12. 32, 12. 33, 12. 34, 12. 35, 12. 36, 12. 37, 12. 38, 12. 39, 12. 40, 12. 41, 12. 42, 12. 43, 12. 44, 12. 45, 12. 46, 12. 47, 12. 48, 12. 49, 12. 50, 12. 51, 12. 52, 12. 53, 12. 54, 12. 55, 12. 56, 12. 57, 12. 58, 12. 59, 12. 60, 12. 61, 12. 62, 12. 63, 12. 64, 12. 65, 12. 66, 12. 67, 12. 68, 12. 69, 12. 70, 12. 71, 12. 72, 12. 73, 12. 74, 12. 75, 12. 76, 12. 77, 12. 78, 12. 79, 12. 80, 12. 81, 12. 82, 12. 83, 12. 84, 12. 85, 12. 86, 12. 87, 12. 88, 12. 89, 12. 90, 12. 91, 12. 92, 12. 93, 12. 94, 12. 95, 12. 96, 12. 97, 12. 98, 12. 99, 12. 100, 12. 101, 12. 102, 12. 103, 12. 104, 12. 105, 12. 106, 12. 107, 12. 108, 12. 109, 12. 110, 12. 111, 12. 112, 12. 113, 12. 114, 12. 115, 12. 116, 12. 117, 12. 118, 12. 119, 12. 120, 12. 121, 12. 122, 12. 123, 12. 124, 12. 125, 12. 126, 12. 127, 12. 128, 12. 129, 12. 130, 12. 131, 12. 132, 12. 133, 12. 134, 12. 135, 12. 136; 13. 14, 13. 15, 13. 16, 13. 17, 13. 18, 13. 19, 13. 20, 13. 21, 13. 22, 13. 23, 13. 24, 13. 25, 13. 26, 13. 27, 13. 28; 13. 29, 13. 30, 13. 31, 13. 32, 13. 33, 13. 34, 13. 35, 13. 36, 13. 37, 13. 38, 13. 39, 13. 40, 13. 41, 13. 42, 13. 43, 13. 44, 13. 45, 13. 46, 13. 47, 13. 48, 13. 49, 13. 50, 13. 51, 13. 52, 13. 53, 13. 54, 13. 55, 13. 56, 13. 57, 13. 58, 13. 59, 13. 60, 13. 61, 13. 62, 13. 63, 13. 64, 13. 65, 13. 66, 13. 67, 13. 68, 13. 69, 13. 70, 13. 71, 13. 72, 13. 73, 13. 74, 13. 75, 13. 76, 13. 77, 13. 78, 13. 79, 13. 80, 13. 81, 13. 82, 13. 83, 13. 84, 13. 85, 13. 86, 13. 87, 13. 88, 13. 89, 13. 90, 13. 91, 13. 92, 13. 93, 13. 94, 13. 95, 13. 96, 13. 97, 13. 98, 13. 99, 13. 100, 13. 101, 13. 102, 13. 103, 13. 104, 13. 105, 13. 106, 13. 107, 13. 108, 13. 109, 13. 110, 13. 111, 13. 112, 13. 113, 13. 114, 13. 115, 13. 116, 13. 117, 13. 118, 13. 119, 13. 120, 13. 121, 13. 122, 13. 123, 13. 124, 13. 125, 13. 126, 13. 127, 13. 128, 13. 129, 13. 130, 13. 131, 13. 132, 13. 133, 13. 134, 13. 135, 13. 136; 14. 15, 14. 16, 14. 17, 14. 18, 14. 19, 14. 20, 14. 21, 14. 22, 14. 23, 14. 24, 14. 25, 14. 26, 14. 27, 14. 28; 14. 29, 14. 30, 14. 31, 14. 32, 14. 33, 14. 34, 14. 35, 14. 36, 14. 37, 14. 38, 14. 39, 14. 40, 14. 41, 14. 42, 14. 43, 14. 44, 14. 45, 14. 46, 14. 47, 14. 48, 14. 49, 14. 50, 14. 51, 14. 52, 14. 53, 14. 54, 14. 55, 14. 56, 14. 57, 14. 58, 14. 59, 14. 60, 14. 61, 14. 62, 14. 63, 14. 64, 14. 65, 14. 66, 14. 67, 14. 68, 14. 69, 14. 70, 14. 71, 14. 72, 14. 73, 14. 74, 14. 75, 14. 76, 14. 77, 14. 78, 14. 79, 14. 80, 14. 81, 14. 82, 14. 83, 14. 84, 14. 85, 14. 86, 14. 87, 14. 88, 14. 89, 14. 90, 14. 91, 14. 92, 14. 93, 14. 94, 14. 95, 14. 96, 14. 97, 14. 98, 14. 99, 14. 100, 14. 101, 14. 102, 14. 103, 14. 104, 14. 105, 14. 106, 14. 107, 14. 108, 14. 109, 14. 110, 14. 111, 14. 112, 14. 113, 14. 114, 14. 115, 14. 116, 14. 117, 14. 118, 14. 119, 14. 120, 14. 121, 14. 122, 14. 123, 14. 124, 14. 125, 14. 126, 14. 127, 14. 128, 14. 129, 14. 130, 14. 131, 14. 132, 14. 133, 14. 134, 14. 135, 14. 136; 15. 16, 15. 17, 15. 18, 15. 19, 15. 20, 15. 21, 15. 22, 15. 23, 15. 24, 15. 25, 15. 26, 15. 27, 15. 28; 15. 29, 15. 30, 15. 31, 15. 32, 15. 33, 15. 34, 15. 35, 15. 36, 15. 37, 15. 38, 15. 39, 15. 40, 15. 41, 15. 42, 15. 43, 15. 44, 15. 45, 15. 46, 15. 47, 15. 48, 15. 49, 15. 50, 15. 51, 15. 52, 15. 53, 15. 54, 15. 55, 15. 56, 15. 57, 15. 58, 15. 59, 15. 60, 15. 61, 15. 62, 15. 63, 15. 64, 15. 65, 15. 66, 15. 67, 15. 68, 15. 69, 15. 70, 15. 71, 15. 72, 15. 73, 15. 74, 15. 75, 15. 76, 15. 77, 15. 78, 15. 79, 15. 80, 15. 81, 15. 82, 15. 83, 15. 84, 15. 85, 15. 86, 15. 87, 15. 88, 15. 89, 15. 90, 15. 91, 15. 92, 15. 93, 15. 94, 15. 95, 15. 96, 15. 97, 15. 98, 15. 99, 15. 100, 15. 101, 15. 102, 15. 103, 15. 104, 15. 105, 15. 106, 15. 107, 15. 108, 15. 109, 15. 110, 15. 111, 15. 112, 15. 113, 15. 114, 15. 115, 15. 116, 15. 117, 15. 118, 15. 119, 15. 120, 15. 121, 15. 122, 15. 123, 15. 124, 15. 125, 15. 126, 15. 127, 15. 128, 15. 129, 15. 130, 15. 131, 15. 132, 15. 133, 15. 134, 15. 135, 15. 136; 16. 17, 16. 18, 16. 19, 16. 20, 16. 21, 16. 22, 16. 23, 16. 24, 16. 25, 16. 26, 16. 27, 16. 28; 16. 29, 16. 30, 16. 31, 16. 32, 16. 33, 16. 34, 16. 35, 16. 36, 16. 37, 16. 38, 16. 39, 16. 40, 16. 41, 16. 42, 16. 43, 16. 44, 16. 45, 16. 46, 16. 47, 16. 48, 16. 49, 16. 50, 16. 51, 16. 52, 16. 53, 16. 54, 16. 55, 16. 56, 16. 57, 16. 58, 16. 59, 16. 60, 16. 61, 16. 62, 16. 63, 16. 64, 16. 65, 16. 66, 16. 67, 16. 68, 16. 69, 16. 70, 16. 71, 16. 72, 16. 73, 16. 74, 16. 75, 16. 76, 16. 77, 16. 78, 16. 79, 16. 80, 16. 81, 16. 82, 16. 83, 16. 84, 16. 85, 16. 86, 16. 87, 16. 88, 16. 89, 16. 90, 16. 91, 16. 92, 16. 93, 16. 94, 16. 95, 16. 96, 16. 97, 16. 98, 16. 99, 16. 100, 16. 101, 16. 102, 16. 103, 16. 104, 16. 105, 16. 106, 16. 107, 16. 108, 16. 109, 16. 110, 16. 111, 16. 112, 16. 113, 16. 114, 16. 115, 16. 116, 16. 117, 16. 118, 16. 119, 16. 120, 16. 121, 16. 122, 16. 123, 16. 124, 16. 125, 16. 126, 16. 127, 16. 128, 16. 129, 16. 130, 16. 131, 16. 132, 16. 133, 16. 134, 16. 135, 16. 136; 17. 18, 17. 19, 17. 20, 17. 21, 17. 22, 17. 23, 17. 24, 17. 25, 17. 26, 17. 27, 17. 28; 17. 29, 17. 30, 17. 31, 17. 32, 17. 33, 17. 34, 17. 35, 17. 36, 17. 37, 17. 38, 17. 39, 17. 40, 17. 41, 17. 42, 17. 43, 17. 44, 17. 45, 17. 46, 17. 47, 17. 48, 17. 49, 17. 50, 17. 51, 17. 52, 17. 53, 17. 54, 17. 55, 17. 56, 17. 57, 17. 58, 17. 59, 17. 60, 17. 61, 17. 62, 17. 63, 17. 64, 17. 65, 17. 66, 17. 67, 17. 68, 17. 69, 17. 70, 17. 71, 17. 72, 17. 73, 17. 74, 17. 75, 17. 76, 17. 77, 17. 78, 17. 79, 17. 80, 17. 81, 17. 82, 17. 83, 17. 84, 17. 85, 17. 86, 17. 87, 17. 88, 17. 89, 17. 90, 17. 91, 17. 92, 17. 93, 17. 94, 17. 95, 17. 96, 17. 97, 17. 98, 17. 99, 17. 100, 17. 101, 17. 102, 17. 103, 17. 104, 17. 105, 17. 106, 17. 107, 17. 108, 17. 109, 17. 110, 17. 111, 17. 112, 17. 113, 17. 114, 17. 115, 17. 116, 17. 117, 17. 118, 17. 119, 17. 120, 17. 121, 17. 122, 17. 123, 17. 124, 17. 125, 17. 126, 17. 127, 17. 128, 17. 129, 17. 130, 17. 131, 17. 132, 17. 133, 17. 134, 17. 135, 17. 136; 18. 19, 18. 20, 18. 21, 18. 22, 18. 23, 18. 24, 18. 25, 18. 26, 18. 27, 18. 28; 18. 29, 18. 30, 18. 31, 18. 32, 18. 33, 18. 34, 18. 35, 18. 36, 18. 37, 18. 38, 18. 39, 18. 40, 18. 41, 18. 42, 18. 43, 18. 44, 18. 45, 18. 46, 18. 47, 18. 48, 18. 49, 18. 50, 18. 51, 18. 52, 18. 53, 18. 54, 18. 55, 18. 56, 18. 57, 18. 58, 18. 59, 18. 60, 18. 61, 18. 62, 18. 63, 18. 64, 18. 65, 18. 66, 18. 67, 18. 68, 18. 69, 18. 70, 18. 71, 18. 72, 18. 73, 18. 74, 18. 75, 18. 76, 18. 77, 18. 78, 18. 79, 18. 80, 18. 81, 18. 82, 18. 83, 18. 84, 18. 85, 18. 86, 18. 87, 18. 88, 18. 89, 18. 90, 18. 91, 18. 92, 18. 93, 18. 94, 18. 95, 18. 96, 18. 97, 18. 98, 18. 99, 18. 100, 18. 101, 18. 102, 18. 103, 18. 104, 18. 105, 18. 106, 18. 107, 18. 108, 18. 109, 18. 110, 18. 111, 18. 112, 18. 113, 18. 114, 18. 115, 18. 116, 18. 117, 18. 118, 18. 119, 18. 120, 18. 121, 18. 122, 18. 123, 18. 124, 18. 125, 18. 126, 18. 127, 18. 128, 18. 129, 18. 130, 18. 131, 18. 132, 18. 133, 18. 134, 18. 135, 18. 136; 19. 20, 19. 21, 19. 22, 19. 23, 19. 24, 19. 25, 19. 26, 19. 27, 19. 28; 19. 29, 19. 30, 19. 31, 19. 32, 19. 33, 19. 34, 19. 35, 19. 36, 19. 37, 19. 38, 19. 39, 19. 40, 19. 41, 19. 42, 19. 43, 19. 44, 19. 45, 19. 46, 19. 47, 19. 48, 19. 49, 19. 50, 19. 51, 19. 52, 19. 53, 19. 54, 19. 55, 19. 56, 19. 57, 19. 58, 19. 59, 19. 60, 19. 61, 19. 62, 19. 63, 19. 64, 19. 65, 19. 66, 19. 67, 19. 68, 19. 69, 19. 70, 19. 71, 19. 72, 19. 73, 19. 74, 19. 75, 19. 76, 19. 77, 19. 78, 19. 79, 19. 80, 19. 81, 19. 82, 19. 83, 19. 84, 19. 85, 19. 86, 19. 87, 19. 88, 19. 89, 19. 90, 19. 91, 19. 92, 19. 93, 19. 94, 19. 95, 19. 96, 19. 97, 19. 98, 19. 99, 19. 100, 19. 101, 19. 102, 19. 103, 19. 104, 19. 105, 19. 106, 19. 107, 19. 108, 19. 109, 19. 110, 19. 111, 19. 112, 19. 113, 19. 114, 19. 115, 19. 116, 19. 117, 19. 118, 19. 119, 19. 120, 19. 121, 19. 122, 19. 123, 19. 124, 19. 125, 19. 126, 19. 127, 19. 128, 19. 129, 19. 130, 19. 131, 19. 132, 19. 133, 19. 134, 19. 135, 19. 136; 20. 21, 20. 22, 20. 23, 20. 24, 20. 25, 20. 26, 20. 27, 20. 28; 20. 29, 20. 30, 20. 31, 20. 32, 20. 33, 20. 34, 20. 35, 20. 36, 20. 37, 20. 38, 20. 39, 20. 40, 20. 41, 20. 42, 20. 43, 20. 44, 20. 45, 20. 46, 20. 47, 20. 48, 20. 49, 20. 50, 20. 51, 20. 52, 20. 53, 20. 54, 20. 55, 20. 56, 20. 57, 20. 58, 20. 59, 20. 60, 20. 61, 20. 62, 20. 63, 20. 64, 20. 65, 20. 66, 20. 67, 20. 68, 20. 69, 20. 70, 20. 71, 20. 72, 20. 73, 20. 74, 20. 75, 20. 76, 20. 77, 20. 78, 20. 79, 20. 80, 20. 81, 20. 82, 20. 83, 20. 84, 20. 85, 20. 86, 20. 87, 20. 88, 20. 89, 20. 90, 20. 91, 20. 92, 20. 93, 20. 94, 20. 95, 20. 96, 20. 97, 20. 98, 20. 99, 20. 100, 20. 101, 20. 102, 20. 103, 20. 104, 20. 105, 20. 106, 20. 107, 20. 108, 20. 109, 20. 110, 20. 111, 20. 112, 20. 113, 20. 114, 20. 115, 20. 116, 20. 117, 20. 118, 20. 119, 20. 120, 20. 121, 20. 122, 20. 123, 20. 124, 20. 125, 20. 126, 20. 127, 20. 128, 20. 129, 20. 130, 20. 131, 20. 132, 20. 133, 20. 134, 20. 135, 20. 136; 21. 22, 21. 23, 21. 24, 21. 25, 21. 26, 21. 27, 21. 28; 21. 29, 21. 30, 21. 31, 21. 32, 21. 33, 21. 34, 21. 35, 21. 36, 21. 37, 21. 38, 21. 39, 21. 40, 21. 41, 21. 42, 21. 43, 21. 44, 21. 45, 21. 46, 21. 47, 21. 48, 21. 49, 21. 50, 21. 51, 21. 52, 21. 53, 21. 54, 21. 55, 21. 56, 21. 57, 21. 58, 21. 59, 21. 60, 21. 61, 21. 62, 21. 63, 21. 64, 21. 65, 21. 66, 21. 67, 21. 68, 21. 69, 21. 70, 21. 71, 21. 72, 21. 73, 21. 74, 21. 75, 21. 76, 21. 77, 21. 78, 21. 79, 21. 80, 21. 81, 21. 82, 21. 83, 21. 84, 21. 85, 21. 86, 21. 87, 21. 88, 21. 89, 21. 90, 21. 91, 21. 92, 21. 93, 21. 94, 21. 95, 21. 96, 21. 97, 21. 98, 21. 99, 21. 100, 21. 101, 21. 102, 21. 103, 21. 104, 21. 105, 21. 106, 21. 107, 21. 108, 21. 109, 21. 110, 21. 111, 21. 112, 21. 113, 21. 114, 21. 115, 21. 116, 21. 117, 21. 118, 21. 119, 21. 120, 21. 121, 21. 122, 21. 123, 21. 124, 21. 125, 21. 126, 21. 127, 21. 128, 21. 129, 21. 130, 21. 131, 21. 132, 21. 133, 21. 134, 21. 135, 21. 136; 22. 23, 22. 24, 22. 25, 22. 26, 22. 27, 22. 28; 22. 29, 22. 30, 22. 31, 22. 32, 22. 33, 22. 34, 22. 35, 22. 36, 22. 37, 22. 38, 22. 39, 22. 40, 22. 41, 22. 42, 22. 43, 22. 44, 22. 45, 22. 46, 22. 47, 22. 48, 22. 49, 22. 50, 22. 51, 22. 52, 22. 53, 22. 54, 22. 55, 22. 56, 22. 57, 22. 58, 22. 59, 22. 60, 22. 61, 22. 62, 22. 63, 22. 64, 22. 65, 22. 66, 22. 67, 22. 68, 22. 69, 22. 70, 22. 71, 22. 72, 22. 73, 22. 74, 22. 75, 22. 76, 22. 77, 22. 78, 22. 79, 22. 80, 22. 81, 22. 82, 22. 83, 22. 84, 22. 85, 22. 86, 22. 87, 22. 88, 22. 89, 22. 90, 22. 91, 22. 92, 22. 93, 22. 94, 22. 95, 22. 96, 22. 97, 22. 98, 22. 99, 22. 100, 22. 101, 22. 102, 22. 103, 22. 104, 22. 105, 22. 106, 22. 107, 22. 108, 22. 109, 22. 110, 22. 111, 22. 112, 22. 113, 22. 114, 22. 115, 22. 116, 22. 117, 22. 118, 22. 119, 22. 120, 22. 121, 22. 122, 22. 123, 22. 124, 22. 125, 22. 126, 22. 127, 22. 128, 22. 129, 22. 130, 22. 131, 22. 132, 22. 133, 22. 134, 22. 135, 22. 136; 23. 24, 23. 25, 23. 26, 23. 27, 23. 28; 23. 29, 23. 30, 23. 31, 23. 32, 23. 33, 23. 34, 23. 35, 23. 36, 23. 37, 23. 38, 23. 39, 23. 40, 23. 41, 23. 42, 23. 43, 23. 44, 23. 45, 23. 46, 23. 47, 23. 48, 23. 49, 23. 50, 23. 51, 23. 52, 23. 53, 23. 54, 23. 55, 23. 56, 23. 57, 23. 58, 23. 59, 23. 60, 23. 61, 23. 62, 23. 63, 23. 64, 23. 65, 23. 66, 23. 67, 23. 68, 23. 69, 23. 70, 23. 71, 23. 72, 23. 73, 23. 74, 23. 75, 23. 76, 23. 77, 23. 78, 23. 79, 23. 80, 23. 81, 23. 82, 23. 83, 23. 84, 23. 85, 23. 86, 23. 87, 23. 88, 23. 89, 23. 90, 23. 91, 23. 92, 23. 93, 23. 94, 23. 95, 23. 96, 23. 97, 23. 98, 23. 99, 23. 100, 23. 101, 23. 102, 23. 103, 23. 104, 23. 105, 23. 106, 23. 107, 23. 108, 23. 109, 23. 110, 23. 111, 23. 112, 23. 113, 23. 114, 23. 115, 23. 116, 23. 117, 23. 118, 23. 119, 23. 120, 23. 121, 23. 122, 23. 123, 23. 124, 23. 125, 23. 126, 23. 127, 23. 128, 23. 129, 23. 130, 23. 131, 23. 132, 23. 133, 23. 134, 23. 135, 23. 136; 24. 25, 24. 26, 24. 27, 24. 28; 24. 29, 24. 30, 24. 31, 24. 32, 24. 33, 24. 34, 24. 35, 24. 36, 24. 37, 24. 38, 24. 39, 24. 40, 24. 41, 24. 42, 24. 43, 24. 44, 24. 45, 24. 46, 24. 47, 24. 48, 24. 49, 24. 50, 24. 51, 24. 52, 24. 53, 24. 54, 24. 55, 24. 56, 24. 57, 24. 58, 24. 59, 24. 60, 24. 61, 24. 62, 24. 63, 24. 64, 24. 65, 24. 66, 24. 67, 24. 68, 24. 69, 24. 70, 24. 71, 24. 72, 24. 73, 24. 74, 24. 75, 24. 76, 24. 77, 24. 78, 24. 79, 24. 80, 24. 81, 24. 82, 24. 83, 24. 84, 24. 85, 24. 86, 24. 87, 24. 88, 24. 89, 24. 90, 24. 91, 24. 92, 24. 93, 24. 94, 24. 95, 24. 96, 24. 97, 24. 98, 24. 99, 24. 100, 24. 101, 24. 102, 24. 103, 24. 104, 24. 105, 24. 106, 24. 107, 24. 108, 24. 109, 24. 110, 24. 111, 24. 112, 24. 113, 24. 114, 24. 115, 24. 116, 24. 117, 24. 118, 24. 119, 24. 120, 24. 121, 24. 122, 24. 123, 24. 124, 24. 125, 24. 126, 24. 127, 24. 128, 24. 129, 24. 130, 24. 131, 24. 132, 24. 133, 24. 134, 24. 135, 24. 136; 25. 26, 25. 27, 25. 28; 25. 29, 25. 30, 25. 31, 25. 32, 25. 33, 25. 34, 25. 35, 25. 36, 25. 37, 25. 38, 25. 39, 25. 40, 25. 41, 25. 42, 25. 43, 25. 44, 25. 45, 25. 46, 25. 47, 25. 48, 25. 49, 25. 50, 25. 51, 25. 52, 25. 53, 25. 54, 25. 55, 25. 56, 25. 57, 25. 58, 25. 59, 25. 60, 25. 61, 25. 62, 25. 63, 25. 64, 25. 65, 25. 66, 25. 67, 25. 68, 25. 69, 25. 70, 25. 71, 25. 72, 25. 73, 25. 74, 25. 75, 25. 76, 25. 77, 25. 78, 25. 79, 25. 80, 25. 81, 25. 82, 25. 83, 25. 84, 25. 85, 25. 86, 25. 87, 25. 88, 25. 89, 25. 90, 25. 91, 25. 92, 25. 93, 25. 94, 25. 95, 25. 96, 25. 97, 25. 98, 25. 99, 25. 100, 25. 101, 25. 102, 25. 103, 25. 104, 25. 105, 25. 106, 25. 107, 25. 108, 25. 109, 25. 110, 25. 111, 25. 112, 25. 113, 25. 114, 25. 115, 25. 116, 25. 117, 25. 118, 25. 119, 25. 120, 25. 121, 25. 122, 25. 123, 25. 124, 25. 125, 26. 126, 26. 127, 26. 128, 26. 129, 26. 130, 26. 131, 26. 132, 26. 133, 26. 134, 26. 135, 26. 136; 26. 27, 26. 28; 26. 29, 26. 30, 26. 31, 26. 32, 26. 33, 26. 34, 26. 35, 26. 36, 26. 37, 26. 38, 26. 39, 26. 40, 26. 41, 26. 42, 26. 43, 26. 44, 26. 45, 26. 46, 26. 47, 26. 48, 26. 49, 26. 50, 26. 51, 26. 52, 26. 53, 26. 54, 26. 55, 26. 56, 26. 57, 26. 58, 26. 59, 26. 60, 26. 61, 26. 62, 26. 63, 26. 64, 26. 65, 26. 66, 26. 67, 26. 68, 26. 69, 26. 70, 26. 71, 26. 72, 26. 73, 26. 74, 26. 75, 26. 76, 26. 77, 26. 78, 26. 79, 26. 80, 26. 81, 26. 82, 26. 83, 26. 84, 26. 85, 26. 86, 26. 87, 26. 88, 26. 89, 26. 90, 26. 91, 26. 92, 26. 93, 26. 94, 26. 95, 26. 96, 26. 97, 26. 98, 26. 99, 26. 100, 26. 101, 26. 102, 26. 103, 26. 104, 26. 105, 26. 106, 26. 107, 26. 108, 26. 109, 26. 110, 26. 111, 26. 112, 26. 113, 26. 114, 26. 115, 26. 116, 26. 117, 26. 118, 26. 119, 26. 120, 26. 121, 26. 122, 26. 123, 26. 124, 26. 125, 26. 126, 26. 127, 26. 128, 26. 129, 26. 130, 26. 131, 26. 132, 26. 133, 26. 134, 26. 135, 26. 136; 27. 28; 27. 29, 27. 30, 27. 31, 27. 32, 27. 33, 27. 34, 27. 35, 27. 36, 27. 37, 27. 38, 27. 39, 27. 40, 27. 41, 27. 42, 27. 43, 27. 44, 27. 45, 27. 46, 27. 47, 27. 48, 27. 49, 27. 50, 27. 51, 27. 52, 27. 53, 27. 54, 27. 55, 27. 56, 27. 57, 27. 58, 27. 59, 27. 60, 27. 61, 27. 62, 27. 63, 27. 64, 27. 65, 27. 66, 27. 67, 27. 68, 27. 69, 27. 70, 27. 71, 27. 72, 27. 73, 27. 74, 27. 75, 27. 76, 27. 77, 27. 78, 27. 79, 27. 80, 27. 81, 27. 82, 27. 83, 27. 84, 27. 85, 27. 86, 27. 87, 27. 88, 27. 89, 27. 90, 27. 91, 27. 92, 27. 93, 27. 94, 27. 95, 27. 96, 27. 97, 27. 98, 27. 99, 27. 100, 27. 101, 27. 102, 27. 103, 27. 104, 27. 105, 27. 106, 27. 107, 27. 108, 27. 109, 27. 110, 27. 111, 27. 112, 27. 113, 27. 114, 27. 115, 27. 116, 27. 117, 27. 118, 27. 119, 27. 120, 27. 121, 27. 122, 27. 123, 27. 124, 27. 125, 27. 126, 27. 127, 27. 128, 27. 129, 27. 130, 27. 131, 27. 132, 27. 133, 27. 134, 27. 135, 27. 136; 28. 29, 28. 30, 28. 31, 28. 32, 28. 33, 28. 34, 28. 35, 28. 36, 28. 37, 28. 38, 28. 39, 28. 40, 28. 41, 28. 42, 28. 43, 28. 44, 28. 45, 28. 46, 28. 47, 28. 48, 28. 49, 28. 50, 28. 51, 28. 52, 28. 53, 28. 54, 28. 55, 28. 56, 28. 57, 28. 58, 28. 59, 28. 60, 28. 61, 28. 62, 28. 63, 28. 64, 28. 65, 28. 66, 28. 67, 28. 68, 28. 69, 28. 70, 28. 71, 28. 72, 28. 73, 28. 74, 28. 75, 28. 76, 28. 77, 28. 78, 28. 79, 28. 80, 28. 81, 28. 82, 28. 83, 28. 84, 28. 85, 28. 86, 28. 87, 28. 88, 28. 89, 28. 90, 28. 91, 28. 92, 28. 93, 28. 94, 28. 95, 28. 96, 28. 97, 28. 98, 28. 99, 28. 100, 28. 101, 28. 102, 28. 103, 28. 104, 28. 105, 28. 106, 28. 107, 28. 108, 28. 109, 28. 110, 28. 111, 28. 112, 28. 113, 28. 114, 28. 115, 28. 116, 28. 117, 28. 118, 28. 119, 28. 120, 28. 121, 28. 122, 28. 123, 28. 124, 28. 125, 28. 126, 28. 127, 28. 128, 28. 129, 28. 130, 28. 131, 28. 132, 28. 133, 28. 134, 28. 135, 28. 136; 29. 30, 29. 31, 29. 32, 29. 33, 29. 34, 29. 35, 29. 36, 29. 37, 29. 38, 29. 39, 29. 40, 29. 41, 29. 42, 29. 43, 29. 44, 29. 45, 29. 46, 29. 47, 29. 48, 29. 49, 29. 50, 29. 51, 29. 52, 29. 53, 29. 54, 29. 55, 29. 56, 29. 57, 29. 58, 29. 59, 29. 60, 29. 61, 29. 62, 29. 63, 29. 64, 29. 65, 29. 66, 29. 67, 29. 68, 29. 69, 29. 70, 29. 71, 29. 72, 29. 73, 29. 74, 29. 75, 29. 76, 29. 77, 29. 78, 29. 79, 29. 80, 29. 81, 29. 82, 29. 83, 29. 84, 29. 85, 29. 86, 29. 87, 29. 88, 29. 89, 29. 90, 29. 91, 29. 92, 29. 93, 29. 94, 29. 95, 29. 96, 29. 97, 29. 98, 29. 99, 29. 100, 29. 101, 29. 102, 29. 103, 29. 104, 29. 105, 29. 106, 29. 107, 29. 108, 29. 109, 29. 110, 29. 111, 29. 112, 29. 113, 29. 114, 29. 115, 29. 116, 29. 117, 29. 118, 29. 119, 29. 120, 29. 121, 29. 122, 29. 123, 29. 124, 29. 125, 29. 126, 29. 127, 29. 128, 29. 129, 29. 130, 29. 131, 29. 132, 29. 133, 29. 134, 29. 135, 29. 136; 30. 31, 30. 32, 30. 33, 30. 34, 30. 35, 30. 36, 30. 37, 30. 38, 30. 39, 30. 40, 30. 41, 30. 42, 30. 43, 30. 44, 30. 45, 30. 46, 30. 47, 30. 48, 30. 49, 30. 50, 30. 51, 30. 52, 30. 53, 30. 54, 30. 55, 30. 56, 30. 57, 30. 58, 30. 59, 30. 60, 30. 61, 30. 62, 30. 63, 30. 64, 30. 65, 30. 66, 30. 67, 30. 68, 30. 69, 30. 70, 30. 71, 30. 72, 30. 73, 30. 74, 30. 75, 30. 76, 30. 77, 30. 78, 30. 79, 30. 80, 30. 81, 30. 82, 30. 83, 30. 84, 30. 85, 30. 86, 30. 87, 30. 88, 30. 89, 30. 90, 30. 91, 30. 92, 30. 93, 30. 94, 30. 95, 30. 96, 30. 97, 30. 98, 30. 99, 30. 100, 30. 101, 30. 102, 30. 103, 30. 104, 30. 105, 30. 106, 30. 107, 30. 108, 30. 109, 30. 110, 30. 111, 30. 112, 30. 113, 30. 114, 30. 115, 30. 116, 30. 117, 30. 118, 30. 119, 30. 120, 30. 121, 30. 122, 30. 123, 30. 124, 30. 125, 30. 126, 30. 127, 30. 128, 30. 129, 30. 130, 30. 131, 30. 132, 30. 133, 30. 134, 30. 135, 30. 136; 31. 32, 31. 33, 31. 34, 31. 35, 31. 36, 31. 37, 31. 38, 31. 39, 31. 40, 31. 41, 31. 42, 31. 43, 31. 44, 31. 45, 31. 46, 31. 47, 31. 48, 31. 49, 31. 50, 31. 51, 31. 52, 31. 53, 31. 54, 31. 55, 31. 56, 31. 57, 31. 58, 31. 59, 31. 60, 31. 61, 31. 62, 31. 63, 31. 64, 31. 65, 31. 66, 31. 67, 31. 68, 31. 69, 31. 70, 31. 71, 31. 72, 31. 73, 31. 74, 31. 75, 31. 76, 31. 77, 31. 78, 31. 79, 31. 80, 31. 81, 31. 82, 31. 83, 31. 84, 31. 85, 31. 86, 31. 87, 31. 88, 31. 89, 31. 90, 31. 91, 31. 92, 31. 93, 31. 94, 31. 95, 31. 96, 31. 97, 31. 98, 31. 99, 31. 100, 31. 101, 31. 102, 31. 103, 31. 104, 31. 105, 31. 106, 31. 107, 31. 108, 31. 109, 31. 110, 31. 111, 31. 112, 31. 113, 31. 114, 31. 115, 31. 116, 31. 117, 31. 118, 31. 119, 31. 120, 31. 121, 31. 122, 31. 123, 31. 124, 31. 125, 31. 126, 31. 127, 31. 128, 31. 129, 31. 130, 31. 131, 31. 132, 31. 133, 31. 134, 31. 135, 31. 136; 32. 33, 32. 34, 32. 35, 32. 36, 32. 37, 32. 38, 32. 39, 32. 40, 32. 41, 32. 42, 32. 43, 32. 44, 32. 45, 32. 46, 32. 47, 32. 48, 32. 49, 32. 50, 32. 51, 32. 52, 32. 53, 32. 54, 32. 55, 32. 56, 32. 57, 32. 58, 32. 59, 32. 60, 32. 61, 32. 62, 32. 63, 32. 64, 32. 65, 32. 66, 32. 67, 32. 68, 32. 69, 32. 70, 32. 71, 32. 72, 32. 73, 32. 74, 32. 75, 32. 76, 32. 77, 32. 78, 32. 79, 32. 80, 32. 81, 32. 82, 32. 83, 32. 84, 32. 85, 32. 86, 32. 87, 32. 88, 32. 89, 32. 90, 32. 91, 32. 92, 32. 93, 32. 94, 32. 95, 32. 96, 32. 97, 32. 98, 32. 99, 32. 100, 32. 101, 32. 102, 32. 103, 32. 104, 32. 105, 32. 106, 32. 107, 32. 108, 32. 109, 32. 110, 32. 111, 32. 112, 32. 113, 32. 114, 32. 115, 32. 116, 32. 117, 32. 118, 32. 119, 32. 120, 32. 121, 32. 122, 32. 123, 32. 124, 32. 125, 32. 126, 32. 127, 32. 128, 32. 129, 32. 130, 32. 131, 32. 132, 32. 133, 32. 134, 32. 135, 32. 136; 33. 34, 33. 35, 33. 36, 33. 37, 33. 38, 33. 39, 33. 40, 33. 41, 33. 42, 33. 43, 33. 44, 33. 45, 33. 46, 33. 47, 33. 48, 33. 49, 33. 50, 33. 51, 33. 52, 33. 53, 33. 54, 33. 55, 33. 56, 33. 57, 33. 58, 33. 59, 33. 60, 33. 61, 33. 62, 33. 63, 33. 64, 33. 65, 33. 66, 33. 67, 33. 68, 33. 69, 33. 70, 33. 71, 33. 72, 33. 73, 33. 74, 33. 75, 33. 76, 33. 77, 33. 78, 33. 79, 33. 80, 33. 81, 33. 82, 33. 83, 33. 84, 33. 85, 33. 86, 33. 87, 33. 88, 33. 89, 33. 90, 33. 91, 33. 92, 33. 93, 33. 94, 33. 95, 33. 96, 33. 97, 33. 98, 33. 99, 33. 100, 33. 101, 33. 102, 33. 103, 33. 104, 33. 105, 33. 106, 33. 107, 33. 108, 33. 109, 33. 110, 33. 111, 33. 112, 33. 113, 33. 114, 33. 115, 33. 116, 33. 117, 33. 118, 33. 119, 33. 120, 33. 121, 33. 122, 33. 123, 33. 124, 33. 125, 33. 126, 33. 127, 33. 128, 33. 129, 33. 130, 33. 131, 33. 132, 33. 133, 33. 134, 33. 135, 33. 136; 34. 35, 34. 36, 34. 37, 34. 38, 34. 39, 34. 40, 34. 41, 34. 42, 34. 43, 34. 44, 34. 45, 34. 46, 34. 47, 34. 48, 34. 49, 34. 50, 34. 51, 34. 52, 34. 53, 34. 54, 34. 55, 34. 56, 34. 57, 34. 58, 34. 59, 34. 60, 34. 61, 34. 62, 34. 63, 34. 64, 34. 65, 34. 66, 34. 67, 34. 68, 34. 69, 34. 70, 34. 71, 34. 72, 34. 73, 34. 74, 34. 75, 34. 76, 34. 77, 34. 78, 34. 79, 34. 80, 34. 81, 34. 82, 34. 83, 34. 84, 34. 85, 34. 86, 34. 87, 34. 88, 34. 89, 34. 90, 34. 91, 34. 92, 34. 93, 34. 94, 34. 95, 34. 96, 34. 97, 34. 98, 34. 99, 34. 100, 34. 101, 34. 102, 34. 103, 34. 104, 34. 105, 34. 106, 34. 107, 34. 108, 34. 109, 34. 110, 34. 111, 34. 112, 34. 113, 34. 114, 34. 115, 34. 116, 34. 117, 34. 118, 34. 119, 34. 120, 34. 121, 34. 122, 34. 123, 34. 124, 34. 125, 34. 126, 34. 127, 34. 128, 34. 129, 34. 130, 34. 131, 34. 132, 34. 133, 34. 134, 34. 135, 34. 136; 35. 36, 35. 37, 35. 38, 35. 39, 35. 40, 35. 41, 35. 42, 35. 43, 35. 44, 35. 45, 35. 46, 35. 47, 35. 48, 35. 49, 35. 50, 35. 51, 35. 52, 35. 53, 35. 54, 35. 55, 35. 56, 35. 57, 35. 58, 35. 59, 35. 60, 35. 61, 35. 62, 35. 63, 35. 64, 35. 65, 35. 66, 35. 67, 35. 68, 35. 69, 35. 70, 35. 71, 35. 72, 35. 73, 35. 74, 35. 75, 35. 76, 35. 77, 35. 78, 35. 79, 35. 80, 35. 81, 35. 82, 35. 83, 35. 84, 35. 85, 35. 86, 35. 87, 35. 88, 35. 89, 35. 90, 35. 91, 35. 92, 35. 93, 35. 94, 35. 95, 35. 96, 35. 97, 35. 98, 35. 99, 35. 100, 35. 101, 35. 102, 35. 103, 35. 104, 35. 105, 35. 106, 35. 107, 35. 108, 35. 109, 35. 110, 35. 111, 35. 112, 35. 113, 35. 114, 35. 115, 35. 116, 35. 117, 35. 118, 35. 119, 35. 120, 35. 121, 35. 122, 35. 123, 35. 124, 35. 125, 35. 126, 35. 127, 35. 128, 35. 129, 35. 130, 35. 131, 35. 132, 35. 133, 35. 134, 35. 135, 35. 136; 36. 37, 36. 38, 36. 39, 36. 40, 36. 41, 36. 42, 36. 43, 36. 44, 36. 45, 36. 46, 36. 47, 36. 48, 36. 49, 36. 50, 36. 51, 36. 52, 36. 53, 36. 54, 36. 55, 36. 56, 36. 57, 36. 58, 36. 59, 36. 60, 36. 61, 36. 62, 36. 63, 36. 64, 36. 65, 36. 66, 36. 67, 36. 68, 36. 69, 36. 70, 36. 71, 36. 72, 36. 73, 36. 74, 36. 75, 36. 76, 36. 77, 36. 78, 36. 79, 36. 80, 36. 81, 36. 82, 36. 83, 36. 84, 36. 85, 36. 86, 36. 87, 36. 88, 36. 89, 36. 90, 36. 91, 36. 92, 36. 93, 36. 94, 36. 95, 36. 96, 36. 97, 36. 98, 36. 99, 36. 100, 36. 101, 36. 102, 36. 103, 36. 104, 36. 105, 36. 106, 36. 107, 36. 108, 36. 109, 36. 110, 36. 111, 36. 112, 36. 113, 36. 114, 36. 115, 36. 116, 36. 117, 36. 118, 36. 119, 36. 120, 36. 121, 36. 122, 36. 123, 36. 124, 36. 125, 36. 126, 36. 127, 36. 128, 36. 129, 36. 130, 36. 131, 36. 132, 36. 133, 36. 134, 36. 135, 36. 136; 37. 38, 37. 39, 37. 40, 37. 41, 37. 42, 37. 43, 37. 44, 37. 45, 37. 46, 37. 47, 37. 48, 37. 49, 37. 50, 37. 51, 37. 52, 37. 53, 37. 54, 37. 55, 37. 56, 37. 57, 37. 58, 37. 59, 37. 60, 37. 61, 37. 62, 37. 63, 37. 64, 37. 65, 37. 66, 37. 67, 37. 68, 37. 69, 37. 70, 37. 71, 37. 72, 37. 73, 37. 74, 37. 75, 37. 76, 37. 77, 37. 78, 37. 79, 37. 80, 37. 81, 37. 82, 37. 83, 37. 84, 37. 85, 37. 86, 37. 87, 37. 88, 37. 89, 37. 90, 37. 91, 37. 92, 37. 93, 37. 94, 37. 95, 37. 96, 37. 97, 37. 98, 37. 99, 37. 100, 37. 101, 37. 102, 37. 103, 37. 104, 37. 105, 37. 106, 37. 107, 37. 108, 37. 109, 37. 110, 37. 111, 37. 112, 37. 113, 37. 114, 37. 115, 37. 116, 37. 117, 37. 118, 37. 119, 37. 120, 37. 121, 37. 122, 37. 123, 37. 124, 37. 125, 37. 126, 37. 127, 37. 128, 37. 129, 37. 130, 37. 131, 37. 132, 37. 133, 37. 134, 37. 135, 37. 136; 38. 39, 38. 40, 38. 41, 38. 42, 38. 43, 38. 44, 38. 45, 38. 46, 38. 47, 38. 48, 38. 49, 38. 50, 38. 51, 38. 52, 38. 53, 38. 54, 38. 55, 38. 56, 38. 57, 38. 58, 38. 59, 38. 60, 38. 61, 38. 62, 38. 63, 38. 64, 38. 65, 38. 66, 38. 67, 38. 68, 38. 69, 38. 70, 38. 71, 38. 72, 38. 73, 38. 74, 38. 75, 38. 76, 38. 77, 38. 78, 38. 79, 38. 80, 38. 81, 38. 82, 38. 83, 38. 84, 38. 85, 38. 86, 38. 87, 38. 88, 38. 89, 38. 90, 38. 91, 38. 92, 38. 93, 38. 94, 38. 95, 38. 96, 38. 97, 38. 98, 38. 99, 38. 100, 38. 101, 38. 102, 38. 103, 38. 104, 38. 105, 38. 106, 38. 107, 38. 108, 38. 109, 38. 110, 38. 111, 38. 112, 38. 113, 38. 114, 38. 115, 38. 116, 38. 117, 38. 118, 38. 119, 38. 120, 38. 121, 38. 122, 38. 123, 38. 124, 38. 125, 38. 126, 38. 127, 38. 128, 38. 129, 38. 130, 38. 131, 38. 132, 38. 133, 38. 134, 38. 135, 38. 136; 39. 40, 39. 41, 39. 42, 39. 43, 39. 44, 39. 45, 39. 46, 39. 47, 39. 48, 39. 49, 39. 50, 39. 51, 39. 52, 39. 53, 39. 54, 39. 55, 39. 56, 39. 57, 39. 58, 39. 59, 39. 60, 39. 61, 39. 62, 39. 63, 39. 64, 39. 65, 39. 66, 39. 67, 39. 68, 39. 69, 39. 70, 39. 71, 39. 72, 39. 73, 39. 74, 39. 75, 39. 76, 39. 77, 39. 78, 39. 79, 39. 80, 39. 81, 39. 82, 39. 83, 39. 84, 39. 85, 39. 86, 39. 87, 39. 88, 39. 89, 39. 90, 39. 91, 39. 92, 39. 93, 39. 94, 39. 95, 39. 96, 39. 97, 39. 98, 39. 99, 39. 100, 39. 101, 39. 102, 39. 103, 39. 104, 39. 105, 39. 106, 39. 107, 39. 108, 39. 109, 39. 110, 39. 111, 39. 112, 39. 113, 39. 114, 39. 115, 39. 116, 39. 117, 39. 118, 39. 119, 39. 120, 39. 121, 39. 122, 39. 123, 39. 124, 39. 125, 39. 126, 39. 127, 39. 128, 39. 129, 39. 130, 39. 131, 39. 132, 39. 133, 39. 134, 39. 135, 39. 136; 40. 41, 40. 42, 40. 43, 40. 44, 40. 45, 40. 46, 40. 47, 40. 48, 40. 49, 40. 50, 40. 51, 40. 52, 40. 53, 40. 54, 40. 55, 40. 56, 40. 57, 40. 58, 40. 59, 40. 60, 40. 61, 40. 62, 40. 63, 40. 64, 40. 65, 40. 66, 40. 67, 40. 68, 40. 69, 40. 70, 40. 71, 40. 72, 40. 73, 40. 74, 40. 75, 40. 76, 40. 77, 40. 78, 40. 79, 40. 80, 40. 81, 40. 82, 40. 83, 40. 84, 40. 85, 40. 86, 40. 87, 40. 88, 40. 89, 40. 90, 40. 91, 40. 92, 40. 93, 40. 94, 40. 95, 40. 96, 40. 97, 40. 98, 40. 99, 40. 100, 40. 101, 40. 102, 40. 103, 40. 104, 40. 105, 40. 106, 40. 107, 40. 108, 40. 109, 40. 110, 40. 111, 40. 112, 40. 113, 40. 114, 40. 115, 40. 116, 40. 117, 40. 118, 40. 119, 40. 120, 40. 121, 40. 122, 40. 123, 40. 124, 40. 125, 40. 126, 40. 127, 40. 128, 40. 129, 40. 130, 40. 131, 40. 132, 40. 133, 40. 134, 40. 135, 40. 136; 41. 42, 41. 43, 41. 44, 41. 45, 41. 46, 41. 47, 41. 48, 41. 49, 41. 50, 41. 51, 41. 52, 41. 53, 41. 54, 41. 55, 41. 56, 41. 57, 41. 58, 41. 59, 41. 60, 41. 61, 41. 62, 41. 63, 41. 64, 41. 65, 41. 66, 41. 67, 41. 68, 41. 69, 41. 70, 41. 71, 41. 72, 41. 73, 41. 74, 41. 75, 41. 76, 41. 77, 41. 78, 41. 79, 41. 80, 41. 81, 41. 82, 41. 83, 41. 84, 41. 85, 41. 86, 41. 87, 41. 88, 41. 89, 41. 90, 41. 91, 41. 92, 41. 93, 41. 94, 41. 95, 41. 96, 41. 97, 41. 98, 41. 99, 41. 100, 41. 101, 41. 102, 41. 103, 41. 104, 41. 105, 41. 106, 41. 107, 41. 108, 41. 109, 41. 110, 41. 111, 41. 112, 41. 113, 41. 114, 41. 115, 41. 116, 41. 117, 41. 118, 41. 119, 41. 120, 41. 121, 41. 122, 41. 123, 41. 124, 41. 125, 41. 126, 41. 127, 41. 128, 41. 129, 41. 130, 41. 131, 41. 132, 41. 133, 41. 134, 41. 135, 41. 136; 42. 43, 42. 44, 42. 45, 42. 46, 42. 47, 42. 48, 42. 49, 42. 50, 42. 51, 42. 52, 42. 53, 42. 54, 42. 55, 42. 56, 42. 57, 42. 58, 42. 59, 42. 60, 42. 61, 42. 62, 42. 63, 42. 64, 42. 65, 42. 66, 42. 67, 42. 68, 42. 69, 42. 70, 42. 71, 42. 72, 42. 73, 42. 74, 42. 75, 42. 76, 42. 77, 42. 78, 42. 79, 42. 80, 42. 81, 42. 82, 42. 83, 42. 84, 42. 85, 42. 86, 42. 87, 42. 88, 42. 89, 42. 90, 42. 91, 42. 92, 42. 93, 42. 94, 42. 95, 42. 96, 42. 97, 42. 98, 42. 99, 42. 100, 42. 101, 42. 102, 42. 103, 42. 104, 42. 105, 42. 106, 42. 107, 42. 108, 42. 109, 42. 110, 42. 111, 42. 112, 42. 113, 42. 114, 42. 115, 42. 116, 42. 117, 42. 118, 42. 119, 42. 120, 42. 121, 42. 122, 42. 123, 42. 124, 42. 125, 42. 126, 42. 127, 42. 128, 42. 129, 42. 130, 42. 131, 42. 132, 42. 133, 42. 134, 42. 135, 42. 136; 43. 44, 43. 45, 43. 46, 43. 47, 43. 48, 43. 49, 43. 50, 43. 51, 43. 52, 43. 53, 43. 54, 43. 55, 43. 56, 43. 57, 43. 58, 43. 59, 43. 60, 43. 61, 43. 62, 43. 63, 43. 64, 43. 65, 43. 66, 43. 67, 43. 68, 43. 69, 43. 70, 43. 71, 43. 72, 43. 73, 43. 74, 43. 75, 43. 76, 43. 77, 43. 78, 43. 79, 43. 80, 43. 81, 43. 82, 43. 83, 43. 84, 43. 85, 43. 86, 43. 87, 43. 88, 43. 89, 43. 90, 43. 91, 43. 92, 43. 93, 43. 94, 43. 95, 43. 96, 43. 97, 43. 98, 43. 99, 43. 100, 43. 101, 43. 102, 43. 103, 43. 104, 43. 105, 43. 106, 43. 107, 43. 108, 43. 109, 43. 110, 43. 111, 43. 112, 43. 113, 43. 114, 43. 115, 43. 116, 43. 117, 43. 118, 43. 119, 43. 120, 43. 121, 43. 122, 43. 123, 43. 124, 43. 125, 43. 126, 43. 127, 43. 128, 43. 129, 43. 130, 43. 131, 43. 132, 43. 133, 43. 134, 43. 135, 43. 136; 44. 45, 44. 46, 44. 47, 44. 48, 44. 49, 44. 50, 44. 51, 44. 52, 44. 53, 44. 54, 44. 55, 44. 56, 44. 57, 44. 58, 44. 59, 44. 60, 44. 61, 44. 62, 44. 63, 44. 64, 44. 65, 44. 66, 44. 67, 44. 68, 44. 69, 44. 70, 44. 71, 44. 72, 44. 73, 44. 74, 44. 75, 44. 76, 44. 77, 44. 78, 44. 79, 44. 80, 44. 81, 44. 82, 44. 83, 44. 84, 44. 85, 44. 86, 44. 87, 44. 88, 44. 89, 44. 90, 44. 91, 44. 92, 44. 93, 44. 94, 44. 95, 44. 96, 44. 97, 44. 98, 44. 99, 44. 100, 44. 101, 44. 102, 44. 103, 44. 104, 44. 105, 44. 106, 44. 107, 44. 108, 44. 109, 44. 110, 44. 111, 44. 112, 44. 113, 44. 114, 44. 115, 44. 116, 44. 117, 44. 118, 44. 119, 44. 120, 44. 121, 44. 122, 44. 123, 44. 124, 44. 125, 44. 126, 44. 127, 44. 128, 44. 129, 44. 130, 44. 131, 44. 132, 44. 133, 44. 134, 44. 135, 44. 136; 45. 46, 45. 47, 45. 48, 45. 49, 45. 50, 45. 51, 45. 52, 45. 53, 45. 54, 45. 55, 45. 56, 45. 57, 45. 58, 45. 59, 45. 60, 45. 61, 45. 62, 45. 63, 45. 64, 45. 65, 45. 66, 45. 67, 45. 68, 45. 69, 45. 70, 45. 71, 45. 72, 45. 73, 45. 74, 45. 75, 45. 76, 45. 77, 45. 78, 45. 79, 45. 80, 45. 81, 45. 82, 45. 83, 45. 84, 45. 85, 45. 86, 45. 87, 45. 88, 45. 89, 45. 90, 45. 91, 45. 92, 45. 93, 45. 94, 45. 95, 45. 96, 45. 97, 45. 98, 45. 99, 45. 100, 45. 101, 45. 102, 45. 103, 45. 104, 45. 105, 45. 106, 45. 107, 45. 108, 45. 109, 45. 110, 45. 111, 45. 112, 45. 113, 45. 114, 45. 115, 45. 116, 45. 117, 45. 118, 45. 119, 45. 120, 45. 121, 45. 122, 45. 1 2 3 4 5. 124, 45. 125, 45. 126, 45. 127, 45. 128, 45. 129, 45. 130, 45. 131, 45. 132, 45. 133, 45. 134, 45. 135, 45. 136; 46. 47, 46. 48, 46. 49, 46. 50, 46. 51, 46. 52, 46. 53, 46. 54, 46. 55, 46. 56, 46. 57, 46. 58, 46. 59, 46. 60, 46. 61, 46. 62, 46. 63, 46. 64, 46. 65, 46. 66, 46. 67, 46. 68, 46. 69, 46. 70, 46. 71, 46. 72, 46. 73, 46. 74, 46. 75, 46. 76, 46. 77, 46. 78, 46. 79, 46. 80, 46. 81, 46. 82, 46. 83, 46. 84, 46. 85, 46. 86, 46. 87, 46. 88, 46. 89, 46. 90, 46. 91, 46. 92, 46. 93, 46. 94, 46. 95, 46. 96, 46. 97, 46. 98, 46. 99, 46. 100, 46. 101, 46. 102, 46. 103, 46. 104, 46. 105, 46. 106, 46. 107, 46. 108, 46. 109, 46. 110, 46. 111, 46. 112, 46. 113, 46. 114, 46. 115, 46. 116, 46. 117, 46. 118, 46. 119, 46. 120, 46. 121, 46. 122, 46. 123, 46. 124, 46. 125, 46. 126, 46. 127, 46. 128, 46. 129, 46. 130, 46. 131, 46. 132, 46. 133, 46. 134, 46. 135, 46. 136; 47. 48, 47. 49, 47. 50, 47. 51, 47. 52, 47. 53, 47. 54, 47. 55, 47. 56, 47. 57, 47. 58, 47. 59, 47. 60, 47. 61, 47. 62, 47. 63, 47. 64, 47. 65, 47. 66, 47. 67, 47. 68, 47. 69, 47. 70, 47. 71, 47. 72, 47. 73, 47. 74, 47. 75, 47. 76, 47. 77, 47. 78, 47. 79, 47. 80, 47. 81, 47. 82, 47. 83, 47. 84, 47. 85, 47. 86, 47. 87, 47. 88, 47. 89, 47. 90, 47. 91, 47. 92, 47. 93, 47. 94, 47. 95, 47. 96, 47. 97, 47. 98, 47. 99, 47. 100, 47. 101, 47. 102, 47. 103, 47. 104, 47. 105, 47. 106, 47. 107, 47. 108, 47. 109, 47. 110, 47. 111, 47. 112, 47. 113, 47. 114, 47. 115, 47. 116, 47. 117, 47. 118, 47. 119, 47. 120, 47. 121, 47. 122, 47. 123, 47. 124, 47. 125, 47. 126, 47. 127, 47. 128, 47. 129, 47. 130, 47. 131, 47. 132, 47. 133, 47. 134, 47. 135, 47. 136; 48. 49, 48. 50, 48. 51, 48. 52, 48. 53, 48. 54, 48. 55, 48. 56, 48. 57, 48. 58, 48. 59, 48. 60, 48. 61, 48. 62, 48. 63, 48. 64, 48. 65, 48. 66, 48. 67, 48. 68, 48. 69, 48. 70, 48. 71, 48. 72, 48. 73, 48. 74, 48. 75, 48. 76, 48. 77, 48. 78, 48. 79, 48. 80, 48. 81, 48. 82, 48. 83, 48. 84, 48. 85, 48. 86, 48. 87, 48. 88, 48. 89, 48. 90, 48. 91, 48. 92, 48. 93, 48. 94, 48. 95, 48. 96, 48. 97, 48. 98, 48. 99, 48. 100, 48. 101, 48. 102, 48. 103, 48. 104, 48. 105, 48. 106, 48. 107, 48. 108, 48. 109, 48. 110, 48. 111, 48. 112, 48. 113, 48. 114, 48. 115, 48. 116, 48. 117, 48. 118, 48. 119, 48. 120, 48. 121, 48. 122, 48. 123, 48. 124, 48. 125, 48. 126, 48. 127, 48. 128, 48. 129, 48. 130, 48. 131, 48. 132, 48. 133, 48. 134, 48. 135, 48. 136; 49. 50, 49. 51, 49. 52, 49. 53, 49. 54, 49. 55, 49. 56, 49. 57, 49. 58, 49. 59, 49. 60, 49. 61, 49. 62, 49. 63, 49. 64, 49. 65, 49. 66, 49. 67, 49. 68, 49. 69, 49. 70, 49. 71, 49. 72, 49. 73, 49. 74, 49. 75, 49. 76, 49. 77, 49. 78, 49. 79, 49. 80, 49. 81, 49. 82, 49. 83, 49. 84, 49. 85, 49. 86, 49. 87, 49. 88, 49. 89, 49. 90, 49. 91, 49. 92, 49. 93, 49. 94, 49. 95, 49. 96, 49. 97, 49. 98, 49. 99, 49. 100, 49. 101, 49. 102, 49. 103, 49. 104, 49. 105, 49. 106, 49. 107, 49. 108, 49. 109, 49. 110, 49. 111, 49. 112, 49. 113, 49. 114, 49. 115, 49. 116, 49. 117, 49. 118, 49. 119, 49. 120, 49. 121, 49. 122, 49. 123, 49. 124, 49. 125, 49. 126, 49. 127, 49. 128, 49. 129, 49. 130, 49. 131, 49. 132, 49. 133, 49. 134, 49. 135, 49. 136; 50. 51, 50. 52, 50. 53, 50. 54, 50. 55, 50. 56, 50. 57, 50. 58, 50. 59, 50. 60, 50. 61, 50. 62, 50. 63, 50. 64, 50. 65, 50. 66, 50. 67, 50. 68, 50. 69, 50. 70, 50. 71, 50. 72, 50. 73, 50. 74, 50. 75, 50. 76, 50. 77, 50. 78, 50. 79, 50. 80, 50. 81, 50. 82, 50. 83, 50. 84, 50. 85, 50. 86, 50. 87, 50. 88, 50. 89, 50. 90, 50. 91, 50. 92, 50. 93, 50. 94, 50. 95, 50. 96, 50. 97, 50. 98, 50. 99, 50. 100, 50. 101, 50. 102, 50. 103, 50. 104, 50. 105, 50. 106, 50. 107, 50. 108, 50. 109, 50. 110, 50. 111, 50. 112, 50. 113, 50. 114, 50. 115, 50. 116, 50. 117, 50. 118, 50. 119, 50. 120, 50. 121, 50. 122, 50. 123, 50. 124, 50. 125, 50. 126, 50. 127, 50. 128, 50. 129, 50. 130, 50. 131, 50. 132, 50. 133, 50. 134, 50. 135, 50. 136; 51. 52, 51. 53, 51. 54, 51. 55, 51. 56, 51. 57, 51. 58, 51. 59, 51. 60, 51. 61, 51. 62, 51. 63, 51. 64, 51. 65, 51. 66, 51. 67, 51. 68, 51. 69, 51. 70, 51. 71, 51. 72, 51. 73, 51. 74, 51. 75, 51. 76, 51. 77, 51. 78, 51. 79, 51. 80, 51. 81, 51. 82, 51. 83, 51. 84, 51. 85, 51. 86, 51. 87, 51. 88, 51. 89, 51. 90, 51. 91, 51. 92, 51. 93, 51. 94, 51. 95, 51. 96, 51. 97, 51. 98, 51. 99, 51. 100, 51. 101, 51. 102, 51. 103, 51. 104, 51. 105, 51. 106, 51. 107, 51. 108, 51. 109, 51. 110, 51. 111, 51. 112, 51. 113, 51. 114, 51. 115, 51. 116, 51. 117, 51. 118, 51. 119, 51. 120, 51. 121, 51. 122, 51. 123, 51. 124, 51. 125, 51. 126, 51. 127, 51. 128, 51. 129, 51. 130, 51. 131, 51. 132, 51. 133, 51. 134,

51.135, 51.136; 52.53, 52.54, 52.55, 52.56, 52.57, 52.58, 52.59, 52.60, 52.61, 52.62, 52.63, 52.64, 52.65, 52.66, 52.67, 52.68, 52.69, 52.70, 52.71, 52.72, 52.73, 52.74, 52.75, 52.76, 52.77, 52.78, 52.79, 52.80, 52.81, 52.82, 52.83, 52.84, 52.85, 52.86, 52.87, 52.88, 52.89, 52.90, 52.91, 52.92, 52.93, 52.94, 52.95, 52.96, 52.97, 52.98, 52.99, 52.100, 52.101, 52.102, 52.13, 52.104, 52.105, 52.106, 52.107, 52.108, 52.109, 52.110, 52.111, 52.112, 52.113, 52.114, 52.115, 52.116, 52.117, 52.118, 52.119, 52.120, 52.121, 52.122, 52.123, 52.124, 52.125, 52.126, 52.128, 52.129, 52.130, 52.131, 52.132, 52.133, 52.134, 52.135, 52.136; 53.54, 53.55, 53.56, 53.57, 53.58, 53.59, 53.60, 53.61, 53.62, 53.63, 53.64, 53.65, 53.66, 53.67, 53.68, 53.69, 53.70, 53.71, 53.72, 53.73, 53.74, 53.75, 53.76, 53.77, 53.78, 53.79, 53.80, 53.81, 53.82, 53.83, 53.84, 53.85, 53.86, 53.87, 53.88, 53.89, 53.90, 53.91, 53.92, 53.93, 53.94, 53.95, 53.96, 53.97, 53.98, 53.99, 53.100, 53.101, 53.102, 53.104, 53.105, 53.106, 53.107, 53.108, 53.109, 53.110, 53.111, 53.112, 53.113, 53.114, 53.115, 53.116, 53.117, 53.118, 53.119, 53.120, 53.121, 53.122, 53.123, 53.124, 53.125, 53.126, 53.127, 53.129, 53.130, 53.131, 53.132, 53.133, 53.134, 53.135, 53.136; 54.55, 54.56, 54.57, 54.58, 54.59, 54.60, 54.61, 54.62, 54.63, 54.64, 54.65, 54.66, 54.67, 54.68, 54.69, 54.70, 54.71, 54.72, 54.73, 54.74, 54.75, 54.76, 54.77, 54.78, 54.79, 54.80, 54.81, 54.82, 54.83, 54.84, 54.85, 54.86, 54.87, 54.88, 54.89, 54.90, 54.91, 54.92, 54.93, 54.94, 54.95, 54.96, 54.97, 54.98, 54.99, 54.100, 54.101, 54.102, 54.103, 54.104, 54.105, 54.106, 54.107, 54.108, 54.109, 54.110, 54.111, 54.112, 54.113, 54.114, 54.115, 54.116, 54.117, 54.118, 54.119, 54.120, 54.121, 54.122, 54.123, 54.124, 54.125, 54.126, 54.127, 54.128, 54.129, 54.130, 54.131, 54.132, 54.133, 54.134, 54.135, 54.136; 55.56, 55.57, 55.58, 55.59, 55.60, 55.61, 55.62, 55.63, 55.64, 55.65, 55.66, 55.67, 55.68, 55.69, 55.70, 55.71, 55.72, 55.73, 55.74, 55.81, 55.82, 55.83, 55.84, 55.85, 55.86, 55.87, 55.88, 55.89, 55.90, 55.91, 55.92, 55.93, 55.94, 55.95, 55.96, 55.97, 55.98, 55.99, 55.100, 55.101, 55.102, 55.103, 55.16, 55.107, 55.108, 55.109, 55.110, 55.111, 55.112, 55.113, 55.114, 55.115, 55.116, 55.131, 55.132, 55.133, 55.134, 55.135, 55.136; 56.57, 56.58, 56.59, 56.60, 56.61, 56.62, 56.63, 56.64, 56.65, 56.66, 56.67, 56.68, 56.69, 56.70, 56.71, 56.72, 56.73, 56.74, 56.75, 56.76, 56.77, 56.78, 56.79, 56.80, 56.81, 56.82, 56.83, 56.84, 56.85, 56.86, 56.87, 56.88, 56.89, 56.90, 56.91, 56.92, 56.93, 56.94, 56.95, 56.96, 56.97, 56.98, 56.99, 56.100, 56.101, 56.102, 56.103, 56.104, 56.105, 56.106, 56.107, 56.108, 56.109, 56.110, 56.111, 56.112, 56.113, 56.114, 56.115, 56.116, 56.117, 56.118, 56.119, 56.120, 56.121, 56.122, 56.123, 56.124, 56.125, 56.126, 56.127, 56.128, 56.129, 56.130, 56.131, 56.132, 56.133, 56.134, 56.135, 56.136; 57.58, 57.59, 57.60, 57.61, 57.62, 57.63,57.64,57.65,57.66,57.67,57.68,57.69,57.70,57.71,57.72,57.73,57.74,57.77,57.77,57.78,57.79,57.80,57.81,57.82, 57.83, 57.84, 57.85, 57.86, 57.87, 57.88, 57.89, 57.90, 57.91, 57.92, 57.93, 57.94, 57.95, 57.96, 57.97, 57.98, 57.99, 57.100, 57.101, 57.102, 57.103, 57.104, 57.105, 57.106, 57.107, 57.18, 57.109, 57.110, 57.111, 57.112, 57.113, 57.114, 57.115, 57.116, 57.117, 57.118, 57.119, 57.120, 57.121, 57.122, 57.123, 57.124, 57.125, 57.126, 57.127, 57.128, 57.129, 57.130, 57.131, 57.132, 57.133, 57.134, 57.135, 57.136; 58.69, 58.60, 58.61, 58.62, 58.63, 58.64, 58.65, 58.66, 58.67, 58.68, 58.69, 58.70, 58.71, 58.72, 58.73, 58.74, 58.75, 58.76, 58.77, 58.78, 58.79, 58.80, 58.81, 58.82, 58.83, 58.84, 58.85, 58.86, 58.87, 58.88, 58.89, 58.90, 58.91, 58.92, 58.93, 58.94, 58.95, 58.96, 58.97, 58.98, 58.99, 58.100, 58.101, 58.102, 58.103, 58.104, 58.105, 58.106, 58.107, 58.108, 58.19, 58.110, 58.111, 58.112, 58.113, 58.114, 58.115, 58.116, 58.117, 58.118, 58.119, 58.120, 58.121, 58.122, 58.123, 58.124, 58.125, 58.126, 58.127, 58.128, 58.129, 58.130, 58.131, 58.132, 58.133, 58.134, 58.135, 58.136; 59.60, 59.61, 59.62, 59.63, 59.64, 59.65, 59.66, 59.67, 59.68, 59.69, 59.70, 59.71, 59.72, 59.73, 59.74, 59.75, 59.76, 59.77, 59.78, 59.79, 59.80, 59.81, 59.82, 59.83, 59.84, 59.85, 59.86, 59.87, 59.88, 59.89, 59.90, 59.91, 59.92, 59.93, 59.94, 59.95, 59.96, 59.97, 59.98, 59.99, 59.100, 59.101, 59.102, 59.103, 59.104, 59.105, 59.106, 59.107, 59.108, 59.109, 59.110, 59.111, 59.112, 59.113, 59.114, 59.115, 59.116, 59.117, 59.118, 59.119, 59.120, 59.121, 59.122, 59.123, 59.124, 59.125, 59.126, 59.127, 59.128, 59.129, 59.130, 59.131, 59.132, 59.133, 59.134, 59.135, 59.136; 60.61, 60.62, 60.63, 60.64, 60.65, 60.66, 60.67, 60.68, 60.69, 60.70, 60.71, 60.72, 60.73, 60.74, 60.75, 60.76, 60.77, 60.78, 60.79, 60.80, 60.81, 60.82, 60.83, 60.84, 60.85, 60.106, 60.107, 60.105, 60.106, 60.107, 60.108, 60.109, 60.110, 60.111, 60.112, 60.113, 60.114, 60.115, 60.116, 60.117, 60.118, 60.119, 60.120, 60.121, 60.122, 60.123, 60.124, 60.125, 60.126, 60.127, 60.128, 60.129, 60.130, 60.131, 60.132, 60.133, 60.134, 60.135, 60.136; 61.62, 61.63, 61.64, 61.65, 61.66, 61.67, 61.68, 61.69, 61.70, 61.71, 61.72, 61.73, 61.74, 61.75, 61.76, 61.77, 61.78, 61.79, 61.80, 61.81, 61.82, 61.83, 61.84, 61.85, 61.86, 61.87, 61.88, 61.89, 61.90, 61.91, 61.92, 61.93, 61.94, 61.95, 61.96, 61.97, 61.98, 61.99, 61.100, 61.101, 61.102, 61.103, 61.104, 61.105, 61.106, 61.107, 61.108, 61.109, 61.110, 61.111, 61.112, 61.113, 61.114, 61.115, 61.116, 61.117, 61.118, 61.119, 61.120, 61.121, 61.122, 61.123, 61.124, 61.125, 61.126, 61.127, 61.128, 61.129, 61.130, 61.131, 61.132, 61.133, 61.134, 61.135, 61.136; 62.66, 62.64, 62.65, 62.66, 62.67, 62.68, 62.69, 62.70, 62.71, 62.72, 62.73, 62.74, 62.75, 62.76, 62.77, 62.78, 62.79, 62.80, 62.81, 62.82, 62.83, 62.84, 62.85, 62.86, 62.87, 62.10, 62.10, 62.10, 62.10, 62.10, 62.112, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.10, 62.1, 62.9, 62.9, 62.9, 62.9, 62.9, 62.9, 62.113, 62.114, 62.115, 62.116, 62.117, 62.118, 62.119, 62.120, 62.121, 62.122, 62.123, 62.124, 62.125, 62.126, 62.127, 62.128, 62.129, 62.130, 62.131, 62.132, 62.133, 62.134, 62.135, 62.136; 63.65, 63.66, 63.67, 63.68, 63.69, 63.70, 63.71, 63.72, 63.73, 63.74, 63.75, 63.76, 63.77, 63.78, 63.79, 63.80, 63.81, 63.82, 63.83, 63.84, 63.85, 63.86, 63.87, 63.88, 63.19, 63.92, 63.93, 63.94, 63.95, 63.96, 63.97, 63.98, 63.99, 63.100, 63.101, 63.102, 63.103, 63.104, 63.105, 63.106, 63.107, 63.108, 63.109, 63.110, 63.111, 63.112, 63.113, 63.114, 63.115, 63.116, 63.117, 63.118, 63.119, 63.120, 63.121, 63.122, 63.123, 63.124, 63.125, 63.126, 63.127, 63.128, 63.129, 63.130, 63.131, 63.132, 63.133, 63.134, 63.135, 63.136; 64.65, 64.66, 64.67, 64.68, 64.69, 64.70, 64.71, 64.72, 64.73, 64.74, 64.75, 64.76, 64.77, 64.78, 64.79, 64.80, 64.81, 64.82, 64.83, 64.84, 64.85, 64.86, 64.87, 64.88, 64.89, 64.90, 64.91, 64.92, 64.93, 64.94, 64.95, 64.96, 64.97, 64.98, 64.99, 64.100, 64.101, 64.102, 64.103, 64.104, 64.105, 64.106, 64.107, 64.108, 64.109, 64.110, 64.111, 64.112, 64.113, 64.114, 64.115, 64.116, 64.117, 64.118, 64.119, 64.120, 64.121, 64.122, 64.123, 64.124, 64.125, 64.126, 64.127, 64.128, 64.129, 64.130, 64.131, 64.132, 64.133, 64.134, 64.135, 64.136; 65.66, 65.67, 65.68, 65.69, 65.70, 65.71, 65.72, 65.73, 65.74, 65.75, 65.76, 65.77, 65.78, 65.79, 65.80, 65.81, 65.82, 65.83, 65.84, 65.85, 65.86, 65.87, 65.88, 65.89, 65.90, 65.91, 65.92, 65.93, 65.94, 65.95, 65.96, 65.97, 65.98, 65.99, 65.100, 65.101, 65.102, 65.103, 65.104, 65.105, 65.106, 65.107, 65.108, 65.109, 65.110, 65.111, 65.112, 65.113, 65.114, 65.115, 65.116, 65.117, 65.118, 65.119, 65.120, 65.121, 65.122, 65.123, 65.124, 65.125, 65.126, 65.127, 65.128, 65.129, 65.130, 65.131, 65.132, 65.133, 65.134, 65.135, 65.136; 66.67, 66.68, 66.69, 66.70, 66.71, 66.72, 66.73, 66.74, 66.75, 66.76, 66.77, 66.78, 66.79, 66.80, 66.81, 66.82, 66.83, 66.84, 66.85, 66.86, 66.87, 66.88, 66.89, 66.90, 66.92, 66.93, 66.94, 66.95, 66.96, 66.97, 66.98, 66.99, 66.100, 66.101, 66.102, 66.103, 66.104, 66.105, 66.106, 66.107, 66.108, 66.109, 66.110, 66.111, 66.112, 66.113, 66.114, 66.115, 66.116, 66.117, 66.118, 66.119, 66.120, 66.121, 66.122, 66.123, 66.124, 66.125, 66.126, 66.127, 66.128, 66.129, 66.130, 66.131, 66.132, 66.133, 66.134, 66.135, 66.136; 67.68, 67.69, 67.70, 67.71, 67.72, 67.73, 67.74, 67.75, 67.76, 67.77, 67.78, 67.79, 67.80, 67.81, 67.82, 67.83, 67.84, 67.85, 67.86, 67.87, 67.88, 67.89, 67.90, 67.91, 67.92, 67.93, 67.94, 67.95, 67.96, 67.97, 67.98, 67.99, 67.100, 67.101, 67.102, 67.103, 67.104, 67.105, 67.106, 67.107, 67.108, 67.109, 67.110, 67.111, 67.112, 67.113, 67.114, 67.115, 67.116, 67.117, 67.118, 67.119, 67.120, 67.121, 67.122, 67.123, 67.124, 67.125, 67.126, 67.127, 67.128, 67.129, 67.130, 67.131, 67.132, 67.133, 67.134, 67.135, 67.136; 68.91, 68.90, 68.91, 68.92, 68.93, 68.70, 68.70, 68.71, 68.72, 68.73, 68.74, 68.75, 68.76, 68.77, 68.78, 68.79, 68.80, 68.81, 68.82, 68.83, 68.84, 68.85, 68.86, 68.87, 68.88, 68.94, 68.95, 68.96, 68.97, 68.98, 68.99, 68.100, 68.101, 68.102, 68.103, 68.104, 68.105, 68.106, 68.107, 68.108, 68.109, 68.110, 68.111, 68.112, 68.113, 68.114, 68.115, 68.116, 68.117, 68.118, 68.119, 68.120, 68.121, 68.122, 68.123, 68.124, 68.125, 68.126, 68.127, 68.128, 68.129, 68.130, 68.131, 68.132, 68.133, 68.134, 68.135, 68.136; 69.70, 69.71, 69.72, 69.73, 69.74, 69.75, 69.76, 69.77, 69.78, 69.79, 69.80, 69.81, 69.82, 69.83, 69.84, 69.85, 69.86, 69.87, 69.88, 69.89, 69.90, 69.91, 69.92, 69.93, 69.94, 69.95, 69.96, 69.97, 69.98, 69.99, 69.100, 69.101, 69.102, 69.103, 69.104, 69.105, 69.106, 69.107, 69.108, 69.109, 69.110, 69.111, 69.112, 69.113, 69.114, 69.115, 69.116, 69.117, 69.118, 69.119, 69.120, 69.121, 69.122, 69.123, 69.124, 69.125, 69.126, 69.127, 69.128, 69.129, 69.130, 69.131, 69.132, 69.133, 69.134, 69.135, 69.136; 70.71, 70.72, 70.73, 70.74, 70.75, 70.76, 70.77, 70.78, 70.79, 70.80, 70.81, 70.82, 70.83, 70.84, 70.85, 70.86, 70.87, 70.88, 70.89, 70.90, 70.91, 70.92, 70.93, 70.94, 70.95, 70.96, 70.97, 70.98, 70.99, 70.100, 70.101, 70.102, 70.103, 70.104, 70.105, 70.106, 70.107, 70.108, 70.109, 70.110, 70.111, 70.112, 70.113, 70.114, 70.115, 70.116, 70.117, 70.118, 70.119, 70.120, 70.121, 70.122, 70.123, 70.124, 70.125, 70.126, 70.127, 70.128, 70.129, 70.130, 70.131, 70.132, 70.133, 70.134, 70.135, 70.136; 71.72, 71.73, 71.74, 71.75, 71.76, 71.77, 71.78, 71.79, 71.80, 71.81, 71.82, 71.83, 71.84, 71.85, 71.86, 71.87, 71.88, 71.89, 71.90, 71.91, 71.92, 71.93, 71.94, 71.95, 71.96, 71.97, 71.98, 71.99, 71.100, 71.101, 71.102, 71.103, 71.104, 71.105, 71.106, 71.107, 71.108, 71.109, 71.110, 71.111, 71.112, 71.113, 71.114, 71.115, 71.116, 71.117, 71.118, 71.119, 71.120, 71.121, 71.122, 71.123, 71.124, 71.125, 71.126, 71.127, 71.128, 71.129, 71.130, 71.131, 71.132, 71.133, 71.134, 71.135, 71.136; 72.73 72.74 72.75 72.76 72.77 72.78 72.79 72.80 72.81 72.82 72.83 72.84 72.85 72.86 72.87 72.88 72.89 72.90 72.91 72.92 72.93 72.94 72.95 72.96 72.97, 72.98, 72.99, 72.100, 72.101, 72.102, 72.103, 72.104, 72.105, 72.106, 72.107, 72.108, 72.109, 72.110, 72.111, 72.112, 72.113, 72.114, 72.115, 72.116, 72.117, 72.118, 72.119, 72.120, 72.121, 72.122, 72.123, 72.124, 72.125, 72.126, 72.127, 72.128, 72.129, 72.130, 72.131, 72.132, 72.133, 72.134, 72.135, 72.136; 73.74, 73.75, 73.76, 73.77, 73.78, 73.79, 73.80, 73.81, 73.82, 73.83,73.84,73.85,73.86,73.87,73.88,73.89,73.90,73.91,73.92,73.93,73.94,73.95,73.96,73.97,73.98, 73.100, 73.101, 73.102, 73.103, 73.104, 73.105, 73.106, 73.107, 73.108, 73.109, 73.110, 73.111, 73.112, 73.113, 73.114, 73.115, 73.116, 73.117, 73.118, 73.119, 73.120, 73.121, 73.122, 73.123, 73.124, 73.125, 73.126, 73.127, 73.128, 73.129, 73.130, 73.131, 73.132, 73.133, 73.134, 73.135, 73.136; 74.75, 74.76, 74.77, 74.78, 74.79, 74.80, 74.81, 74.82, 74.83, 74.84, 74.85, 74.86, 74.87, 74.88, 74.89, 74.90, 74.91, 74.92, 74.93, 74.94, 74.95, 74.96, 74.97, 74.98, 74.99, 74.100, 74.101, 74.102, 74.103, 74.104, 74.105, 74.106, 74.107, 74.108, 74.109, 74.110, 74.111, 74.112, 74.113, 74.114, 74.115, 74.116, 74.117, 74.118, 74.119, 74.120, 74.121, 74.122, 74.123, 74.124, 74.125, 74.126, 74.127, 74.128, 74.129, 74.130, 74.131, 74.132, 74.133, 74.134, 74.135, 74.136; 75.96, 75.96, 75.97, 75.98, 75.99, 75.16, 75.75, 75.77, 75.78, 75.79, 75.80, 75.81, 75.82, 75.83, 75.84, 75.85, 75.86, 75.87, 75.88, 75.89, 75.90, 75.101, 75.102, 75.103, 75.104, 75.105, 75.106, 75.107, 75.108, 75.109, 75.110, 75.111, 75.112, 75.113, 75.114, 75.126, 75.127, 75.128, 75.129, 75.130, 75.131, 75.132, 75.133, 75.134, 75.135, 75.136; 76.77, 76.78, 76.79, 76.80, 76.81, 76.82, 76.83, 76.84, 76.85, 76.86, 76.87, 76.88, 76.89, 76.90, 76.91, 76.92, 76.93, 76.94, 76.102, 76.103, 76.104, 76.105, 76.106, 76.107, 76.108, 76.109, 76.110, 76.111, 76.112, 76.113, 76.114, 76.115, 76.116, 76.117, 76.118, 76.119, 76.120, 76.121, 76.122, 76.123, 76.124, 76.125, 76.127, 76.128, 76.129, 76.130, 76.131, 76.132, 76.133, 76.134, 76.135, 76.136; 77.78, 77.79, 77.80, 77.81, 77.82, 77.83, 77.84, 77.85, 77.86, 77.87, 77.88, 77.89, 77.90, 77.91, 77.92, 77.93, 77.94, 77.95, 77.96, 77.97, 77.98, 77.99, 77.100, 77.101, 77.102, 77.103, 77.104, 77.105, 77.106, 77.107, 77.108, 77.109, 77.110, 77.111, 77.112, 77.113, 77.114, 77.115, 77.116, 77.117, 77.118, 77.119, 77.120, 77.121, 77.122, 77.123, 77.124, 77.125, 77.126, 77.127, 77.128, 77.129, 77.130, 77.131, 77.132, 77.133, 77.134, 77.135, 77.136; 78.98, 78.80, 78.81, 78.82, 78.83, 78.84, 78.85, 78.86, 78.87, 78.88, 78.89, 78.90, 78.91, 78.92, 78.93, 78.94, 78.95, 78.96, 78.97, 78.98, 78.99, 78.100, 78.101, 78.102, 78.103, 78.104, 78.105, 78.106, 78.107, 78.108, 78.109, 78.110, 78.111, 78.112, 78.113, 78.114, 78.115, 78.116, 78.117, 78.118, 78.119, 78.120, 78.121, 78.122, 78.123, 78.124, 78.125, 78.126, 78.127, 78.129, 78.130, 78.131, 78.132, 78.133, 78.134, 78.135, 78.136; 79.80, 79.81, 79.82, 79.83, 79.84, 79.85, 79.86, 79.87, 79.88, 79.89, 79.90, 79.91, 79.92, 79.93, 79.94, 79.95, 79.96, 79.97, 79.98, 79.99, 79.100, 79.101, 79.102, 79.103, 79.104, 79.105, 79.106, 79.107, 79.108, 79.109, 79.110, 79.111, 79.112, 79.113, 79.114, 79.115, 79.116, 79.117, 79.118, 79.119, 79.120, 79.121, 79.122, 79.123, 79.124, 79.125, 79.126, 79.127, 79.128, 79.129, 79.130, 79.131, 79.132, 79.133, 79.134, 79.135, 79.136; 80.81, 80.82, 80.83, 80.84, 80.85, 80.86, 80.87, 80.88, 80.89, 80.90, 80.91, 80.92, 80.93, 80.94, 80.95, 80.96, 80.97, 80.98, 80.99, 80.100, 80.101, 80.102, 80.103, 80.104, 80.105, 80.106, 80.107, 80.108, 80.109, 80.110, 80.111, 80.112, 80.113, 80.114, 80.115, 80.116, 80.117, 80.118, 80.119, 80.120, 80.121, 80.122, 80.123, 80.124, 80.125, 80.126, 80.127, 80.128, 80.129, 80.130, 80.131, 80.132, 80.133, 80.134, 80.135, 80.136; 81.82, 81.83, 81.84, 81.85, 81.86, 81.87, 81.88, 81.89, 81.90, 81.91, 81.92, 81.93, 81.94, 81.95, 81.96, 81.97, 81.98, 81.99, 81.100, 81.101, 81.102, 81.103, 81.104, 81.105, 81.106, 81.107, 81.108, 81.109, 81.110, 81.111, 81.112, 81.113, 81.114, 81.115, 81.116, 81.117, 81.118, 81.119, 81.120, 81.121, 81.122, 81.123, 81.124, 81.125, 81.126, 81.127, 81.128, 81.129, 81.130, 81.131, 81.132, 81.133, 81.134, 81.135, 81.136; 82.83, 82.84, 82.85, 82.86, 82.87, 82.88, 82.89, 82.90, 82.91, 82.92, 82.93, 82.94, 82.95, 82.96, 82.97, 82.98, 82.99, 82.100, 82.101, 82.102, 82.103, 82.104, 82.105, 82.106, 82.107, 82.108, 82.109, 82.110, 82.111, 82.112, 82.113, 82.114, 82.115, 82.116, 82.117, 82.118, 82.119, 82.120, 82.121, 82.122, 82.123, 82.124, 82.125, 82.126, 82.127, 82.128, 82.129, 82.130, 82.131, 82.132, 82.133, 82.134, 82.135, 82.136; 83.84, 83.85, 83.86, 83.87, 83.88, 83.89, 83.90, 83.91, 83.92, 83.93, 83.94, 83.95, 83.96, 83.97, 83.98, 83.99, 83.100, 83.101, 83.102, 83.103, 83.104, 83.105, 83.106, 83.107, 83.108, 83.110, 83.110, 83.111, 83.112, 83.113, 83.114, 83.115, 83.116, 83.117, 83.118, 83.119, 83.120, 83.121, 83.122, 83.123, 83.124, 83.125, 83.126, 83.127, 83.128, 83.129, 83.130, 83.131, 83.132, 83.133, 83.134, 83.135, 83.136; 84.85, 84.86, 84.87, 84.88, 84.89, 84.90, 84.91, 84.92, 84.93, 84.94, 84.95, 84.96, 84.97, 84.98, 84.99, 84.100, 84.101, 84.102, 84.103, 84.104, 84.105, 84.106, 84.107, 84.108, 84.110, 84.111, 84.112, 84.113, 84.114, 84.115, 84.116, 84.117, 84.118, 84.119, 84.120, 84.121, 84.122, 84.123, 84.124, 84.125, 84.126, 84.127, 84.128, 84.129, 84.130, 84.131, 84.132, 84.133, 84.134, 84.135, 84.136; 85.86, 85.87, 85.88, 85.89, 85.90, 85.91, 85.92, 85.93, 85.94, 85.95, 85.96, 85.97, 85.98, 85.99, 85.100, 85.101, 85.102, 85.103, 85.104, 85.105, 85.106, 85.107, 85.108, 85.109, 85.110, 85.111, 85.112, 85.113, 85.114, 85.115, 85.116, 85.117, 85.118, 85.119, 85.120, 85.121, 85.122, 85.123, 85.124, 85.125, 85.126, 85.127, 85.128, 85.129, 85.130, 85.131, 85.132, 85.133, 85.134, 85.135, 85.136; 86.87, 86.88, 86.89, 86.90, 86.91, 86.92, 86.93, 86.94, 86.95, 86.96, 86.97, 86.98, 86.99, 86.100, 86.101, 86.102, 86.103, 86.104, 86.105, 86.106, 86.107, 86.108, 86.109, 86.110, 86.112, 86.113, 86.114, 86.115, 86.116, 86.117, 86.118, 86.119, 86.120, 86.121, 86.122, 86.123, 86.124, 86.125, 86.126, 86.127, 86.128, 86.129, 86.130, 86.131, 86.132, 86.133, 86.134, 86.135, 86.136; 87.88, 87.89, 87.90, 87.91, 87.92, 87.93, 87.94, 87.95, 87.96, 87.97, 87.98, 87.99, 87.100, 87.101, 87.102, 87.103, 87.104, 87.105, 87.106, 87.107, 87.108, 87.109, 87.110, 87.111, 87.113, 87.114, 87.115, 87.116, 87.117, 87.118, 87.119, 87.120, 87.121, 87.122, 87.123, 87.124, 87.125, 87.126, 87.127, 87.128, 87.129, 87.130, 87.131, 87.132, 87.133, 87.134, 87.135, 87.136; 88.89, 88.90, 88.91, 88.92, 88.93, 88.94, 88.95, 88.96, 88.97, 88.98, 88.99, 88.100, 88.101, 88.102, 88.103, 88.104, 88.105, 88.106, 88.107, 88.108, 88.109, 88.110, 88.111, 88.112, 88.113, 88.114, 88.115, 88.116, 88.117, 88.118, 88.119, 88.120, 88.121, 88.122, 88.123, 88.124, 88.125, 88.126, 88.127, 88.128, 88.129, 88.130, 88.131, 88.132, 88.133, 88.134, 88.135, 88.136; 89.90, 89.91, 89.92, 89.93, 89.94, 89.95, 89.96, 89.97, 89.98, 89.99, 89.100, 89.101, 89.102, 89.103, 89.104, 89.105, 89.106, 89.107, 89.108, 89.109, 89.110, 89.111, 89.112, 89.113, 89.114, 89.115, 89.116, 89.117, 89.118, 89.119, 89.120, 89.121, 89.122, 89.123, 89.124, 89.125, 89.126, 89.127, 89.128, 89.129, 89.130, 89.131, 89.132, 89.133, 89.134, 89.135, 89.136; 90.91, 90.92, 90.93, 90.94, 90.95, 90.96, 90.97, 90.98, 90.99, 90.100, 90.101, 90.102, 90.103, 90.104, 90.105, 90.106, 90.107, 90.108, 90.109, 90.110, 90.111, 90.112, 90.113, 90.116, 90.117, 90.118, 90.119, 90.120, 90.121, 90.122, 90.123, 90.124, 90.125, 90.126, 90.127, 90.128, 90.129, 90.130, 90.131, 90.132, 90.133, 90.134, 90.135, 90.136; 91.92, 91.93, 91.94, 91.95, 91.96, 91.97, 91.98, 91.99, 91.100, 91.101, 91.102, 91.103, 91.104, 91.105, 91.106, 91.107, 91.108, 91.109, 91.110, 91.111, 91.112, 91.113, 91.114, 91.115, 91.116, 91.117, 91.118, 91.119, 91.120, 91.121, 91.122, 91.123, 91.124, 91.125, 91.126, 91.127, 91.128, 91.129, 91.130, 91.131, 91.132, 91.133, 91.134, 91.135, 91.136; 92.93, 92.94, 92.95, 92.96, 92.97, 92.98, 92.99, 92.100, 92.101, 92.102, 92.103, 92.104, 92.105, 92.106, 92.107, 92.108, 92.109, 92.110, 92.111, 92.112, 92.113, 92.114, 92.115, 92.116, 92.117, 92.118, 92.119, 92.120, 92.121, 92.122, 92.123, 92.124, 92.125, 92.126, 92.127, 92.128, 92.129, 92.130, 92.131, 92.132, 92.133, 92.134, 92.135, 92.136; 93.94, 93.95, 93.96, 93.97, 93.98, 93.99, 93.100, 93.101, 93.102, 93.103, 93.104, 93.105, 93.106, 93.107, 93.108, 93.109, 93.110, 93.111, 93.112, 93.113, 93.114, 93.115, 93.116, 93.117, 93.119, 93.120, 93.121, 93.122, 93.123, 93.124, 93.125, 93.126, 93.127, 93.128, 93.129, 93.130, 93.131, 93.132, 93.133, 93.134, 93.135, 93.136; 94.95, 94.96, 94.97, 94.98, 94.99, 94.100, 94.101, 94.102, 94.103, 94.104, 94.105, 94.106, 94.107, 94.108, 94.109, 94.110, 94.111, 94.112, 94.113, 94.114, 94.115, 94.116, 94.117, 94.118, 94.119, 94.120, 94.121, 94.122, 94.123, 94.124, 94.125, 94.126, 94.127, 94.128, 94.129, 94.130, 94.131, 94.132, 94.133, 94.134, 94.135, 94.136; 95.96, 95.97, 95.98, 95.99, 95.100, 95.101, 95.102,95.103,95.104,95.105,95.106,95.107,95.108,95.109,95.110,95.111,95.112,95.113,95.114,95.115,95.116,95.117,95.118,95.119,95.120, 95.121, 95.122, 95.123, 95.124, 95.125, 95.126, 95.127, 95.128, 95.129, 95.130, 95.131, 95.132, 95.133, 95.134, 95.135, 95.136; 96.97, 96.98, 96.99, 96.100, 96.101, 96.102, 96.103, 96.104, 96.105, 96.106, 96.107, 96.108, 96.109, 96.110, 96.111, 96.112, 96.113, 96.114, 96.115, 96.116, 96.117, 96.118, 96.119, 96.122, 96.123, 96.124, 96.125, 96.126, 96.127, 96.128, 96.129, 96.130, 96.131, 96.132, 96.133, 96.134, 96.135, 96.136; 97.98, 97.99, 97.100, 97.101, 97.102, 97.103, 97.104, 97.105, 97.106, 97.107, 97.108, 97.109, 97.110, 97.111, 97.112, 97.113, 97.114, 97.115, 97.116, 97.117, 97.118, 97.119, 97.120, 97.121, 97.123, 97.124, 97.125, 97.126, 97.127, 97.128, 97.129, 97.130, 97.131, 97.132, 97.133, 97.134, 97.135, 97.136; 98.99, 98.100, 98.101, 98.102, 98.103, 98.104, 98.105, 98.106, 98.107, 98.108, 98.109, 98.110, 98.111, 98.112, 98.113, 98.114, 98.115, 98.116, 98.117, 98.118, 98.119, 98.120, 98.121, 98.124, 98.125, 98.126, 98.127, 98.128, 98.129, 98.130, 98.131, 98.132, 98.133, 98.134, 98.135, 98.136; 99.100, 99.101, 99.102, 99.103, 99.104, 99.105, 99.106, 99.107, 99.108, 99.109, 99.110, 99.111, 99.112, 99.113, 99.114, 99.115, 99.116, 99.117, 99.118, 99.119, 99.120, 99.121, 99.122, 99.123, 99.125, 99.126, 99.127, 99.128, 99.129, 99.130, 99.131, 99.132, 99.133, 99.134, 99.135, 99.136; 100.101, 100.102, 100.103, 100.104, 100.105, 100.106, 100.107, 100.108, 100.109, 100.110, 100.111, 100.112, 100.113, 100.114, 100.115, 100.116, 100.117, 100.118, 100.119, 100.120, 100.121, 100.122, 100.123, 100.124, 100.126, 100.127, 100.128, 100.129, 100.130, 100.131, 100.132, 100.133, 100.134, 100.135, 100.136; 101.102, 101.103, 101.104, 101.105, 101.106, 101.107, 101.108, 101.109, 101.110, 101.111, 101.112, 101.113, 101.114, 101.115, 101.116, 101.117, 101.118, 101.119, 101.120, 101.121, 101.122, 101.123, 101.124, 101.125, 101.126, 101.127, 101.128, 101.129, 101.130, 101.131, 101.132, 101.133, 101.134, 101.135, 101.136; 102.104, 102.104, 102.105, 102.106, 102.107, 102.108, 102.109, 102.110, 102.111, 102.112, 102.113, 102.114, 102.115, 102.116, 102.117, 102.118, 102.119, 102.120, 102.121, 102.122, 102.123, 102.124, 102.125, 102.126, 102.127, 102.128, 102.129, 102.130, 102.131, 102.132, 102.133, 102.134, 102.135, 102.136; 103.104, 103.105, 103.106, 103.107, 103.108, 103.109, 103.110, 103.111, 103.112, 103.113, 103.114, 103.115, 103.116, 103.117, 103.118, 103.119, 103.120, 103.121, 103.122, 103.123, 103.124, 103.125, 103.129, 103.130, 103.131, 103.132, 103.133, 103.134, 103.135, 103.136; 104.105, 104.106, 104.107, 104.108, 104.109, 104.110, 104.111, 104.112, 104.113, 104.114, 104.115, 104.116, 104.117, 104.118, 104.119, 104.120, 104.121, 104.122, 104.123, 104.124, 104.125, 104.126, 104.127, 104.128, 104.130, 104.131, 104.132, 104.133, 104.134, 104.135, 104.136; 105.106, 105.107, 105.108, 105.109, 105.110, 105.111, 105.112, 105.113, 105.114, 105.115, 105.116, 105.117, 105.118, 105.119, 105.120, 105.121, 105.122, 105.123, 105.124, 105.125, 105.126, 105.127, 105.128, 105.129, 105.130, 105.131, 105.132, 105.133, 105.134, 105.135, 105.136; 106.107, 106.108, 106.109, 106.110, 106.111, 106.112, 106.113, 106.114, 106.115, 106.116, 106.117, 106.118, 106.119, 106.120, 106.121, 106.122, 106.123, 106.124, 106.125, 106.126, 106.127, 106.128, 106.129, 106.130, 106.131, 106.132, 106.133, 106.134, 106.135, 106.136; 107.108, 107.109, 107.110, 107.111, 107.112, 107.113, 107.114, 107.115, 107.116, 107.117, 107.118, 107.119, 107.120, 107.121, 107.122, 107.123, 107.124, 107.125, 107.126, 107.127, 107.128, 107.129, 107.130, 107.131, 107.133, 107.134, 107.135, 107.136; 108.109, 108.110, 108.111, 108.112, 108.113, 108.114, 108.115, 108.116, 108.117, 108.118, 108.119, 108.120, 108.121, 108.122, 108.123, 108.124, 108.125, 108.126, 108.127, 108.128, 108.129, 108.130, 108.131, 108.132, 108.133, 108.134, 108.135, 108.136; 109.110, 109.111, 109.112, 109.113, 109.114, 109.115, 109.116, 109.117, 109.118, 109.119, 109.120, 109.121, 109.122, 109.123, 109.124, 109.125, 109.126, 109.127, 109.128, 109.129, 109.130, 109.131, 109.132, 109.133, 109.134, 109.135, 109.136; 110.111, 110.112, 110.113, 110.114, 110.115, 110.116, 110.117, 110.118, 110.119, 110.120, 110.121, 110.122, 110.123, 110.124, 110.125, 110.126, 110.127, 110.128, 110.129, 110.130, 110.131, 110.132, 110.133, 110.134, 110.135, And 110.136;

 An example of a combination of two compounds containing at least one non-cytotoxic compound, using the numerical designators described above in the form #. #, Is described below, which may or may not further include combinations of other compounds:

111.112, 111.113, 111.114, 111.115, 111.116, 111.117, 111.118, 111.119, 111.120, 111.121, 111.122, 111.123, 111.124, 111.125, 111.126, 111.127, 111.128, 111.129, 111.130, 111.131, 111.132, 111.133, 111.134, 111.135, 111.136; 112.113, 112.114, 112.115, 112.116, 112.117, 112.118, 112.119, 112.120, 112.121, 112.122, 112.123, 112.124, 112.125, 112.126, 112.127, 112.128, 112.129, 112.130, 112.131, 112.132, 112.133, 112.134, 112.135, 112.136; 113.114, 113.115, 113.116, 113.117, 113.118, 113.119, 113.120, 113.121, 113.122, 113.123, 113.124, 113.125, 113.126, 113.127, 113.128, 113.129, 113.130, 113.131, 113.132, 113.133, 113.134, 113.135, 113.136; 114.115, 114.116, 114.117, 114.118, 114.119, 114.120, 114.121, 114.122, 114.123, 114.124, 114.125, 114.126, 114.127, 114.128, 114.129, 114.130, 114.131, 114.132, 114.133, 114.134, 114.135, 114.136; 115.116, 115.117, 115.118, 115.119, 115.120, 115.121, 115.122, 115.123, 115.124, 115.125, 115.126, 115.127, 115.128, 115.129, 115.130, 115.131, 115.132, 115.133, 115.134, 115.135, 115.136; 116.117, 116.118, 116.119, 116.120, 116.121, 116.122, 116.123, 116.124, 116.125, 116.126, 116.127, 116.128, 116.129, 116.130, 116.131, 116.132, 116.133, 116.134, 116.135, 116.136; 117.118, 117.119, 117.120, 117.121, 117.122, 117.123, 117.124, 117.125, 117.126, 117.127, 117.128, 117.129, 117.130, 117.131, 117.132, 117.133, 117.134, 117.135, 117.136; 118.119, 118.120, 118.121, 118.122, 118.123, 118.124, 118.125, 118.126, 118.127, 118.128, 118.129, 118.130, 118.131, 118.132, 118.133, 118.134, 118.135, 118.136; 119.120, 119.121, 119.122, 119.123, 119.124, 119.125, 119.126, 119.127, 119.128, 119.129, 119.130, 119.131, 119.132, 119.133, 119.134, 119.135, 119.136; 120.121, 120.122, 120.123, 120.124, 120.125, 120.126, 120.127, 120.128, 120.129, 120.130, 120.131, 120.132, 120.133, 120.134, 120.135, 120.136; 121.122, 121.123, 121.124, 121.125, 121.126, 121.127, 121.128, 121.129, 121.130, 121.131, 121.132, 121.133, 121.134, 121.135, 121.136; 122.123, 122.124, 122.125, 122.126, 122.127, 122.128, 122.129, 122.130, 122.131, 122.132, 122.133, 122.134, 122.135, 122.136; 123.124, 123.125, 123.126, 123.127, 123.128, 123.129, 123.130, 123.131, 123.132, 123.133, 123.134, 123.135, 123.136; 124.125, 124.126, 124.127, 124.128, 124.129, 124.130, 124.131, 124.132, 124.133, 124.134, 124.135, 124.136; 125.126, 125.127, 125.128, 125.129, 125.130, 125.131, 125.132, 125.133, 125.134, 125.135, 125.136; 126.127, 126.128, 126.129, 126.130, 126.131, 126.132, 126.133, 126.134, 126.135, 126.136; 127.128, 127.129, 127.130, 127.131, 127.132, 127.133, 127.134, 127.135, 127.136; 128.129, 128.130, 128.131, 128.132, 128.133, 128.134, 128.135, 128.136; 129.130, 129.131, 129.132, 129.133, 129.134, 129.135, 129.136; 130.131, 130.132, 130.133, 130.134, 130.135, 130.136; 131.132, 131.133, 131.134, 131.135, 131.136; 132.133, 132.134, 132.135, 132.136; 133.134, 133.135, 133.136; 134.135, 134.136; And 135.136.

 As shown in Figure 7, the method of the present invention provides for optimal observation in the dose response curve. In one embodiment, the method of the invention selects a drug concentration for a patient using a dose response curve.

In another embodiment, a drug concentration is selected that induces apoptosis in at least about 75% of the cells in the sample. In another embodiment, a drug concentration is selected that induces apoptosis in at least about 50% of the cells in the sample. In another embodiment, a drug concentration is selected that induces apoptosis in at least about 25% of the cells in the sample. Further, a standard drug concentration, such as the EC50 value of the drug, may not correspond to a given dose for administering a multireflex therapeutic regimen. In another embodiment, the method of the invention selects the drug concentration for the patient using optimal conditions. In another embodiment, the method of the invention selects the drug concentration for the patient using EC50. In another embodiment, the method of the invention selects the drug concentration for the patient using EC90. In another embodiment, the methods of the invention utilize cellular responses of normal cells to select the desired drug composition and concentration for the neoplastic conditions.

 The methods of the invention may also be used to evaluate the pharmacokinetic properties of both cytotoxic and non-cytotoxic drug compositions. As shown in FIG. 8, the pharmacokinetic properties for individual patients differ depending on the different drug compositions. In one embodiment, the methods of the invention determine the kinetic characteristics of the drug for a particular condition. In another embodiment, the methods of the invention determine the kinetic characteristics of the drug composition for a particular condition. In certain embodiments, drug regimens are selected based on the kinetic characteristics of the drug for the particular condition.

 Figure 8 also shows that the method of the present invention is useful for measuring the ability of different drug compositions to induce apoptosis in different time periods. Moreover, the method of the present invention is useful for assessing the difference in the induction of apoptosis between different drug compositions after different time periods have elapsed. In one embodiment, the method detects the induction of apoptosis at about 10, 12, 14, 16, 18, 20, 22, or 24 hours, or in a range defined by any two of the above numbers do. In another embodiment, the method detects induction of apoptosis at about 36 or 48 hours. In another embodiment, the method detects induction of apoptosis at about 72 hours.

 The relevant measurement is the minimum time required to incubate the cells with the drug to effectively induce (e. G. Apoptosis), as shown in Fig. For these measurements, a similar analysis can be achieved by incubating the drug compositions for 15 minutes, washing the drug off, and waiting 48 hours to measure apoptosis. In one embodiment, the method comprises incubating the drug for at least about 30 minutes, 45 minutes, 1 hour, 2 hours, or 4 hours prior to washing, or for any two of the above defined ranges, . In another embodiment, the method detects induction of apoptosis at about 24 or 48 hours. In another embodiment, the method detects the induction of apoptosis at about 72 hours.

 In certain embodiments, an apparatus is provided that can perform the method of the present invention. For example, a plate that already contains the individual drug or combination of drugs at various concentrations may be provided prior to introducing the cell sample. Alternatively, the device into which the cell sample has been previously introduced into the wells can be provided before introduction of the individual drug or drug combination at various concentrations.

 The method of the present invention can be used to leverage a mouse model capable of proliferating and expanding primary human patient cells from serological malignancies (Pearson et al. Curr Top Microbiol Immunol. 2008; 324: 25-51; Ito et al. Curr Top Microbiol Immunol. 2008; 324: 53-76). These mouse models can, for example, expand the number of patient cells available for in vitro testing using the ExviTech platform. This allows considerably more drug and drug combinations to be tested in in vitro patient cells proliferated by these mouse models and allows in vivo testing of the best drug and drug combinations in the same mouse model. In one embodiment, the efficacy and toxicity of the in vitro tested drug compositions in the patient's sample is demonstrated in a mouse model used to proliferate cells taken from the patient. In other embodiments, drug compositions of cytotoxic drugs are tested in in vitro samples of mouse models, and best drug compositions are evaluated in mouse models. In other embodiments, drug compositions of cytotoxic drugs in combination with non-cytotoxic drugs, e. G., Amplifiers and approved drugs, are tested in in vitro samples of mouse models, I will be evaluated. In other embodiments, non-cytotoxic drugs, e.g., drug compositions of both adjuvants and approved drugs, are tested in in vitro samples of mouse models, and the highest drug compositions are assessed in vivo in mouse models.

 Another advantage of the method of the present invention is the ability to create customized reports on patient response to different drug compositions and concentrations. In one embodiment, the method includes generating a report that summarizes the analysis results. In another embodiment, the method comprises providing the patient with the report. In another embodiment, the method comprises providing the report to a person in charge of medical care of the patient. In another embodiment, the method includes providing the report to a person in charge of interpreting the analysis step. In one embodiment, the report includes raw data. In another embodiment, the report comprises a dose response curve. In another embodiment, the report includes a summary of the patient's response to the drug composition and drug concentration.

 Although the exact dosage will be determined on a per drug basis, in most cases, some generalization of the dosage can be made. For example, the dosage of a drug in a human adult patient is, for example, from about 1 mg to about 500 mg per day, or preferably from about 10 mg to about 100 mg per day. The dosage form may be oral, but is preferably intravenous. For example, the compositions of the present invention may be administered by continuous intravenous infusion. Alternatively, in certain embodiments, subcutaneous or intramuscular delivery dosage forms are prepared. The dosage ranges for cytotoxic and non-cytotoxic drugs are generally similar. Any of the pharmaceutical compositions of the present invention include pharmaceutically acceptable salts of the described compounds. The compounds may be administered during a continuous treatment period, e. G., Week, month, or longer. It will also be appreciated by those skilled in the art that the exact formulation, route of administration, and dosage of the drug and drug composition of the present invention may be chosen by a physician in view of the condition of the patient. For example, the amount of drug or drug combination administered will depend upon the individual being treated, the weight of the individual, the severity of the disease, the manner of administration, or the judgment of the prescribing physician.

It will be understood by those skilled in the art that many modifications may be made without departing from the spirit of the invention. Accordingly, the embodiments of the invention disclosed herein are illustrative and are not intended to limit the scope of the invention. Any references referred to herein are fully incorporated by reference in their entirety for the entities discussed herein.

[Example]

Example 1: Detection of flow cytometry in normal and neoplastic cells with apoptosis

 The in vitro therapeutic index can be determined by measuring the power of the drug composition that induces apoptosis. Figures 1 and 2 illustrate the ability to detect apoptotic cells using flow cytometry and to distinguish between normal and tumor phenotypes. In Figure 1, phosphatidylserine expression was detected on apoptotic cells using the reagent annexin V coupled with fluorescein isothiocyanate (FITC). Fluorescence intensity appears on the y-axis and cell size appears on the x-axis. Figure 1 shows the ability to identify apoptotic cells (upper left box) and live cells (lower right group), and using the appropriate combination of monoclonal antibodies and a multivariate analysis strategy, Demonstrating that it is possible to differentiate leukemic cells present in the sample from the remaining normal cells. Figure 2 shows a precursor B-ALL adult event showing BCR / ABL gene rearrangement [t (9; 22) positive]. Two cell subgroups, namely leukemic (pale gray) and normal (charcoal gray), were detected in CD 19 positive cells using multiple monoclonal antibody staining and analyzed by quantitative flow cytometry. Leukemic cells express a unique phenotype associated with dislocation (migration) (co-expression of CD34, but low, with relatively dual CD38 expression).

Example 2: Norms for in vitro evaluation of drug compositions

 An in vitro screening process for drug compositions is illustrated in FIG. The blood sample can be divided into small aliquots and dispensed into well sized plates of the appropriate size. These well plates each contain the individual drug or drug combination at various concentrations. To facilitate optimal method development, samples are diluted in RPMI medium and concentrated to about 20,000 leukemic cells per well. Separately, other aliquots were identified for immunophenotype using flow cytometry for identification of normal and pathological cells and detection of basal apoptosis. (Not shown) in order to confirm the natural level of apoptosis independent of drug therapy.

 After about 48 hours, each well with the sample exposed to the drug was treated with buffer to dissolve the red blood cell population and concentrate the white blood cell population. Next, each well is incubated with Annexin V, and tumor cells and normal cells are accurately detected and confirmed using an antibody combination to detect apoptosis. Flow cytometry can be used to assess the effect of each drug on each cell type and quantify the level of selective cell death induced by each drug.

 Next, new tests can be initiated using additional aliquots to assess the results and identify relevant results, such as concentrations, of the 10 highest drug compositions identified in previous studies. After the assay is performed on a patient sample, selection of the appropriate drug or drug combination to selectively induce apoptosis in neoplastic cells such as leukemia cells can be achieved.

Example 3: Individual patient response demonstrates the cytotoxic effect of various drugs that have been modernly approved for the treatment of chronic lymphocytic leukemia

 Using the methods of the present invention, chloramphenicol, cyclophosphamide, vincristine, mitoxantrone, and doxorubicin (5 drugs approved for chronic lymphocytic leukemia (CLL)) at 30 μM concentration were analyzed in a variety of patients . The results for the efficacy of individually approved cytotoxic drugs to induce apoptosis in malignant cells from nine in vitro patient samples are provided in FIG. Figure 4 shows that there is a high individual-to-person variability in the drug response and therefore indicates the importance of use for the customized medical examinations described herein.

 When defined as inducing less than 60% of apoptosis in a patient sample as measured by annexin V-positive cells, for a patient response to individual drug therapy at a concentration of 30 μM, some drugs are generally poor Has a patient response. Figure 4 also shows that some of the patients (in particular Pl.0105, P2.0019, and P2.035) exhibit excessive ex vivo resistance to in vitro mitoxantrone. Alternatively, in the case of two marked patients, only doxorubicin is highly effective and the other three drugs are resistant, indicating that this type of testing can be very effective in guiding treatment to these patients. Although the results from in vitro assays are more accurate to predict drug tolerance than drug efficacy (eg, as shown in Table 2), if the drug does not kill malignant cells in vitro, the same cells should be killed in vivo Not the same.

Example 4: Induction of apoptosis by cytotoxic and non-cytotoxic drugs in CLL

 The ability of non-cytotoxic drugs to induce apoptosis in in vitro samples was explored using peripheral blood samples obtained from patients with CLL (chronic lymphocytic leukemia). Approximately 900 commercially available drugs were screened one by one in 23 different patient samples in vitro. Figure 6 shows the efficacy of several clinically approved cytotoxic drugs and some non-cytotoxic drugs on serologic neoplasms in these CLL in vitro samples. The results are shown as% apoptosis. As shown in the results, clinically approved drugs induced apoptosis in more than 75% of malignant cells. From left to right, the non-cytotoxic drugs studied are paroxetine, fluoxetine, sertraline, guanabenz, and astemizole. From left to right, the cytotoxic drugs studied were fludarabine, chlorambucil, and mitoxantrone. Figure 6 demonstrates that the non-cytotoxic drugs selectively kill the same malignant cells with an in vitro efficacy similar to that of an approved cytotoxic drug. These unexpected results indicate that the non-cytotoxic drugs have significant therapeutic benefit to the patients studied in FIG.

 Additional demonstration studies have been conducted on the induction of apoptosis by non-cytotoxic drugs. Figure 7 compares the difference between malignant leukemic cells and non-malignant T and NK cells in the cytotoxic effect of paroxetine. At a concentration of approximately 30 μM, paroxetine induced apoptosis in nearly 100% of leukemic cells. However, at the same concentration of about 30 μM, paroxetine induced apoptosis in only 15% of T and NK cells. In conclusion, FIG. 7 shows that paroxetine selectively induces apoptosis in in vitro malignant CLL cells, while non-malignant NK and T cells have minimal effect.

 Non-cytotoxic drugs commonly prescribed in treatment modalities may have a highly selective apoptotic potency against malignant cells. One such case is shown in Figure 12 for CLL patients and is expressed as a percentage of annexin V-positive cells induced by different drugs. A great diversity has been found in the cytotoxic effects of different drugs used in CLL therapy (e.g., vincristine, mitoxantrone, and cyclophosphamide). Surprisingly, two non-cytotoxic compounds normally included to address the side effects caused by chemotherapy (e.g., omeprazole and acyclovir) exhibited apoptosis rates similar to cytotoxic agents. Thus, tailored medical examinations that include non-cytotoxic drugs such as those set forth herein including those exemplified herein may provide unexpected potential therapeutic benefits to the patient. For example, adding non-cytotoxic drugs to such in vitro tests may represent new and unexpected treatments that assist in standard therapy.

Example 5: 24 hour and 48 hour analysis of the ability of selected drugs to induce apoptosis

 The epidemiology of apoptosis induction was evaluated by observing the percentage of cells undergoing apoptosis at 24 and 48 hours. Figure 8 shows that the non-cytotoxic drug sertraline removes malignant CLL cells faster than an approved cytotoxic agent. In Figure 8, a whole blood sample from a patient diagnosed with CLL was analyzed for response to drug treatment. Whole blood samples were incubated with three drugs currently approved for the treatment of sertraline or CLL (fludarabine, chlorambucil, or mitoxantrone). After addition of the drug, the whole blood sample was incubated for 24 or 48 hours before analysis. As shown in the results of FIG. 8, the mechanism of induction of Apoptosis was significantly higher than that of the three clinical approved drugs (inducing about 40%, 45%, and 50% apoptosis after 24 hours, respectively) Lean (more than 90% induction of apoptosis after 24 hours) is faster. Therefore, sertraline induced almost complete apoptosis within 24 hours, whereas other CLL drugs required 48 hours for optimal efficacy.

 The faster in vitro apoptosis may be said to be a higher in vivo efficacy. However, Figure 8 also shows that the efficacy of these four drugs is equal after 48 hours. Figure 8 highlights the usefulness of evaluating multiple incubation times to select optimal treatment for each patient. In addition, Figure 8 shows that several variables must be studied for the development of optimal multiple therapies (e.g., drug composition and incubation time).

Example 6: Differential induction of apoptosis by drugs of the same pharmacological grade

 Paroxetine is an alternative serotonin reuptake inhibitor (SSRI). Other members of the SSRI-rated compounds were tested to determine if the SSRI pharmacological grade of drug had general Apoptosis-inducing properties. Figure 9 summarizes the ability of six SSRIs (paroxetine, fluoxetine, sertraline, citalopram, fluvoxamine, and zimellidin) to induce apoptosis in malignant CLL cells. Of the six drugs, only three (paroxetine, fluoxetine, and sertraline) induced apoptosis similar to clinically-approved cytotoxic drugs, as the results of Figure 9 show. These differences between drugs sharing similar pharmacological properties with similar mechanisms of action demonstrate the need for the in vitro test methodology described in this invention to select among pharmaceutical analogues. These differences also indicate the need for in vitro testing of each drug, regardless of pharmacological rating. These differences also highlight the importance of the ability to study many variables to develop customized medical tests.

Example 7: Frequency of Patients Showing 80% or More Apoptosis Induction by the Same Medication Treatment

 The apoptotic potency of the non-cytotoxic drug is more varied by individual-by-individual than by the apoptotic efficacy of the approved cytotoxic drug (e. G., As shown in Figure 4) ). This change is also shown in FIG. 23 samples from the CLL diagnostic patient (total blood or combination of bone marrow) were screened first with about 2,000 compounds (some samples were insufficient to screen all compounds). The screening measures the ability of each compound to selectively induce apoptosis in a population of leukemic cells of each patient. A compound is considered a "hit" for a particular patient if the compound induces apoptosis in more than 80% of the leukemic population and has little or no effect on the normal cell population.

 The results in Figure 10 show that only a small number of drugs are available (80-100%) on most of the patient samples. Similarly, only 10 additional compounds were effective in 40-60% of patient samples. Forty-five drugs were effective at 40-60% of the samples, 66 drugs were effective at 20-40% of the samples, and 229 additional drugs were effective at less than 20% of the samples. The addition of these drugs means that the 353 drug is effective in inducing in vitro apoptosis in these 23 samples. These were drugs that were not previously discussed as treatments for serologic malignancies, which would require the screening of large numbers of cytotoxic and non-cytotoxic drugs with the development of customized medical tests and determine optimal drug regimens . Such unexpected data may have major clinical implications for the treatment of serological neoplasms

 The regulatory authority permits the use of a small number of non-cytotoxic drugs to alleviate the effects of cytotoxic therapy on patients with serologic neoplasms. Nonetheless, the potential potency observed and required in the present invention for most other non-cytotoxic drugs may soon become part of this therapy as a concept of customized medical advances. The multifunctional custom medical exam described in this invention identifies potentially useful non-cytotoxic drugs for each individual patient, which represents a novel and therapeutically beneficial way that was previously unavailable.

Example 8: Enhancing the efficacy of approved cytotoxic drugs by non-cytotoxic drugs

 As an example of a potential benefit of a customized medical screening test, the compound sertraline identified as being hit for CLL patient samples can enhance the response of chlorambucil. This is shown in FIG. Chlorambucil is the most commonly prescribed drug used in the frontline therapy of CLL in about 25% of patients unable to tolerate fluaravarin-based therapies. As chlorambucil is highly cytotoxic and causes multiple side effects, seeking a way to limit the dose will greatly benefit the patient. In certain trials, sertraline is an antidepressant that is available in generic formulations and sold on the market for several years. At certain concentrations chloramphenicol alone does not induce significant apoptosis (bottom curve), but sub-maximal dose of sertraline improves the level of apoptosis (top curve). Such examples illustrate potential alternative therapies with the ability to improve the response of the prescribed chemotherapeutic treatment.

 These results are consistent with methods of using flow cytometry, which allows exploration of the effects of all drugs approved and possible combinations of drugs and drug combinations, including non-cytotoxic conjugates used to alleviate the side effects of the chemotherapy strategy And the need for customized medical tests using high-throughput screening.

Example 9: Design of a Multi-Therapy Customized Medical (PM) Test for CLL according to the PETHEMA Therapeutic Regimen

 A 96-well plate design for customized medical examinations for patients with CLL (chronic lymphocytic leukemia), without considering non-cytotoxic drugs, is shown in FIGS. 13-15. In each plate, Column 1 contained 0.34% DMSO solution as negative control and Column 12 contained 50 μM paroxetine solution and 50 μM staurosporine solution (well E-H) as a positive control. Drug and drug combinations on plates are those approved for these symptoms in the usual treatment regimens. In each column, wells 2-6 and 7-11 contain a five-point dose response of each of these drugs and drug combinations, with a dilution factor of 2: 3. Columns 2 and 7 thus contain the highest concentration of drug, which is set for each drug according to its therapeutic range. Chlorambucil (CH); Fludarabine (Fl); Mapospamide (MA); Doxorubicin (DO); Binocristine (VI); Prednisolone (Pr); Mitoxantrone (MI); 2-chloro deoxyadenosine (2-CDA); Flavopyridol (FL); Melphalan (ME); Me-Prednisolone (MEPR); Bendamustine (BE); Pentostatin (PE); Rituximab (RIT); Alemtouzam (ALE).

Example 10: Design of PM test for MM according to PETHEMA treatment standard

 A 96-well plate design for customized medical examinations for MM (multiple myeloma) patients, without consideration of non-cytotoxic drugs, is shown in Figures 16-21 in six panels A to F. Plate layouts were generated according to current treatment standards, including individual drugs. In each plate, Column 1 contains 0.34% DMSO solution as negative control and Column 12 contains 50 [mu] M paroxetine and 50 [mu] M staurosporine solution as a positive control (well E-H). Drugs and drug combinations in these plates are those approved for these symptoms in the usual treatment regimens. In each column, wells 2-6 and 7-11 contain a five-point dose response of each of these drugs and drug combinations, with a dilution factor of 2: 3. Columns 2 and 7 thus contain the highest concentration of drug, which is set for each drug according to its therapeutic range. Plates 4-6 also include all possible duplex combinations of approved norms to clarify synergism between all drugs. Dexamethasone (D); Prednisone (P); Melphalan (M); Cyclophosphamide (C); Doxorubicin (A); Vincristine (Vi); Carmustine (BCNU-B); Bortezomib (V); Thalidomide (T); Lanalidomide (L); Panavidostat (Pa); Tanespimycin (Tn); perifosine (Pe); Barrenostat (Vo); Rapamycin (Ra); Everonimus (Ev); Temsirolimus (Te); Ti fipa mip (Ti); Cisplatin (cP); Ethofoside (E).

Example 11: Design of PM test for ALL according to PETHEMA treatment standard

 A 96-well plate design for a customized medical examination for patients with ALL (acute lymphocytic leukemia) is shown in FIG. Plate layouts were generated according to current treatment standards, including individual drugs used in monotherapy. The study design is to determine the ability of the following drugs to induce apoptosis in patient samples: methotrexate (MTX), 6-mercaptopurine (6MP), cytarabine (ARA-C) (DNR), adriamycin, mitoxantrone (M), ethofoside, teniposide (VM-26), cyclophosphamide (CF), ifosfamide (IFOS), vincristine VIND), asparaginase (L-ASA), imatinib (IMAT), rituximab (R), prednisone (P), hydrocortisone (HC), dexamethasone (DXM), lucophorin (Om), ondansetron (O), allopurinol (Allop), and Peel Grass Team (GCSF). The design of these 96-well plates allows a number of chemotherapy strategies to be compared at the same time. The design is also to test the effectiveness of adjuvants and drugs used to alleviate side effects, alone or in combination with monotherapy drugs.

Example 12: Design of PPM test for MDS according to PETHEMA treatment standard

 A 96-well plate design for customized medical examinations for MDS (myelodysplastic syndrome) patients is shown in FIG. Plate layouts were generated according to current treatment standards, including individual drugs used in monotherapy. The study design is to determine the ability of the following drugs to induce apoptosis in patient samples: erythropoietin (EPO), peel glass team (GCSF), thalidomide, cyclosporine (CsA), timoglobin (ATG ), Arsenic trioxide, azacytidine, decitabine, fluda, etoposide (VP-16), cytarabine (ARA-C), dirubicin (Ida), carboplatin Pred, Ondans, Om, Alop, Cotri, and Folic Acid. The design of these 96-well plates allows for the simultaneous comparison of a number of combination therapy strategies. This design also tests the effects of adjuvants and drugs used to alleviate side effects, alone or in combination with monotherapy drugs.

Example 13: Design of a PM test for AML according to the treatment standard

 A 96-well plate design for customized medical examinations for AML (acute myelogenous leukemia, not M3) patients is shown in FIG. Plate layouts were generated according to current treatment standards, including individual drugs used in monotherapy. The study design is to determine the ability of the following drugs to induce apoptosis in a patient sample: dauno, dairubicin (Ida), cytarabine (ARA-C), mitoxan Mitox, Etoposide (VP16), Fluda, Filgas Team (GCSF), Omeprazole (Om), Ondansetron (Ondans), Alopurinol (Alop), Co- , Folic acid (AcF), amsacrine (AMSA), carboplatin liposomal Dauno lipo, gentuzumam oogamycin (GO), and hydroxyurea. The design of these 96-well plates allows for the simultaneous comparison of a number of combination therapy strategies. This design also tests the effects of adjuvants and drugs used to alleviate side effects, alone or in combination with monotherapy drugs.

Example 14: Design of PM test for AML-M3 according to the treatment standard

 A 96-well plate design for customized medical examinations for patients with AML-M3 (acute myelogenous leukemia M3) is shown in FIG. Plate layouts were generated according to current treatment standards, including individual drugs used in monotherapy. The study design is to determine the ability of the following drugs to induce apoptosis in a patient sample: Tretinoin (ATRA), Ida rubicin, Mitoxan, (ARA-C), 6-mercaptopurine (6-MP), methotrexate (MTX), ondansetron (Ondans), allopurinol (Alop), omeprazole (Om), dexamethasone (Dexa), daunorubicin ), Etoposide (VP-16), fludarabine (Fluda), carboplatin (Carbop), liposomal daunorubicin (Dauno lipo), ko-tree pastoris (Cotri), and folate (FAc). The design of these 96-well plates allows for the simultaneous comparison of a number of combination therapy strategies. This design also tests the effects of adjuvants and drugs used to alleviate side effects, alone or in combination with monotherapy drugs.

Example 15: MTT proliferation assay in primary leukemic cell line

TOM-I cells were induced from bone marrow cells of patients with Phi-positive acute lymphocytic leukemia (ALL). MOLT-4 cells were derived from human acute lymphocytic leukemia cell line. Standard MTT assays were performed to determine the IC ₅₀ for individual items to be tested on a particular cell line. The MTT assay is based on the decomposition of the yellow tetrazolium salt, MTT, into purple formazan crystals by metabolically active cells. The formazan is then dissolved and the concentration is determined by optical density at 570 nm. Six to eight different concentrations of sertraline were analyzed at 24 hours after triplicate treatment.

The effect of sertraline on the inhibition of cell proliferation was evaluated at 24 hours in TOM-I and MOLT-4 primary cell lines. The IC ₅₀ for the MOLT-4 cell line was 40 μM and the IC ₅₀ for the TOM-I cell line was 50 μM (FIG. 26).

Example 16: Apoptosis Determination of apoptosis using an ELISA

 One-step sandwich ELISA was performed using the TOM-I and MOLT-4 cells obtained in Example 15. The one-step sandwich ELISA is based on the quantitation of histone-complex DNA fragments (mono- and oligonucleosomes) from the cytoplasm of cells after induction of apoptosis or release from necrotic cells.

 The effects of sertraline on the induction of apoptosis at 24 hours in TOM-I and MOLT-4 primary cell lines were evaluated. Sertraline increased the apoptosis index 7-fold in MOLT-4 cells (Figure 27).

Example 17: Active caspase-3 crystals

 Caspase-3 activity was determined using a Western blot of extracts from two acute lymphocytic cells (TOM-I and MOLT-4) exposed to high concentrations of sertraline. The extracts were taken at 24 and 48 hours. It is a protease that acts as a marker of active caspase-3 apoptosis.

 The effects of sertraline on the induction of active caspase-3 at 24 hours in TOM-I and MOLT-4 primary cell lines were evaluated. Sertraline induces maximal induction of active caspase-3 in MOLT-4 cells at a concentration of 70 μM (FIG. 28).

Example 18: In vitro efficacy of a multifunctional combination in a CLL sample

 Multitotal combinations of rituximab, fludarabine, mitoxantrone, and cyclophosphamide were tested at maximum concentration in CLL samples. Cyclophosphamide was not tested directly, but rather was tested with his metabolite mapposmide (the active compound in the human body). The four major individual drugs were resistant (eg, rituximab, fludarabine, mitoxantrone, and cyclophosphamide) (Figure 29, left). Multireflexion with fludarabine and rituximab was also resistant (Figure 29, center). The triple therapy regimen (eg, fluaravarin and cyclophosphamide; fludarabine, cyclophosphamide, and mitoxantrone; and fluaravarin, cyclophosphamide, and rituximab) , Basically removing all leukemic cells (right) (Fig. 29).

 Fludarabine and cyclophosphamide; Fludarabine, cyclophosphamide, and mitoxantrone; And a 5-point response curve for the combination of fludarabine, cyclophosphamide, and rituximab to characterize the in vitro efficacy (Figure 30-32). These curves show a significant synergistic effect on the drug combination and highlight the importance of the drug combination as described herein.

Example 19: Effect of a single drug at five different concentrations in two patients

 The combination of fludarabine, mapposfamide, and fludarabine and mappospamide was tested at five concentrations in two patients P2.0144 (Figure 33, left) and P2.0149 (Figure 33, right) . Combination index (CI) was calculated using the program Calcusyn (Chou et al., Adv Enzyme Regul 1984; 22: 27-55; Chou et al., Eur J Biochem 1981; 115 (l): 207-16) (Shown at the top of the panel). CI is a quantitative measure of the extent of drug interactions in terms of additive effects. A synergistic effect occurs when CI < 1, an addition effect occurs when CI to I, and an antagonistic effect occurs when CI > The dose-reduction index (DRI) is a measure of how much the dose of each drug in a synergistic combination is reduced compared to the dose of the individual drug alone at a given effect level.

Figure 34 shows a combination index for the fractionation effect based on weight and Talaray method (upper panel). The multiplication table shows the observed values. The black line corresponds to the model simulation. The middle panel shows drug interaction isobolograms based on the weighted and Talaray method at three different levels of response (ED ₅₀ , ED ₇₅ , and ED ₉₀ ) based on dose response estimates. Points drawn on each axis correspond to the estimated dose for this response individually for each drug. The straight line indicates the area of the addition effect on the combination. The points for the combined dose found below this line indicate drug synergism. Figures 33 and 34 show that the combination of fluaravarin and mapposfamide enhances cytotoxicity over single drug efficacy against leukemic CLL B-cells.

Example 20: Effect of incubation time on the efficacy of drugs that induce apoptosis in malignant cells of CLL samples

The effect of incubation time on the efficacy of fluradarin and sertraline in inducing apoptosis in malignant cells of CLL samples was tested. Figure 35 shows the curves for fluaravirus (left panel) and sertraline (right panel), where apoptosis was measured at 24 hours (upper) or 48 hours (lower). In both cases, the drug was incubated with the sample for 30 minutes, 4 hours, or 8 hours, then the drug was washed away and 24 or 48 hours was waited for apoptosis. Cetraline, a non-cytotoxic drug that induces apoptosis in CLL malignant cells, exhibited faster kinetics than fludarabine

Example 21: Number of drug combinations

 The number of 2 drugs, 3 drugs, and 4 drug combinations in 15 drugs was calculated.

 Figure 36 shows the number of two drug combinations that can be made from 15 individual drugs. Each drug is expressed as a number and dark cells as two drug combinations. This represents a total of 105 combinations of the two drugs.

 Figure 37 shows the number of the only 3 drug combinations that can be made from 15 individual drugs. All two drug combinations listed in the top row of each matrix are combined with a single drug in the left column and the central box is painted in light gray. All two drug combinations listed in the bottom row of each matrix are combined with a single drug on the left column and the center box is painted dark gray. This produces a total of 455 unique combinations of the three drugs.

 Figure 38 shows the number of the only four drug combinations that can be made from 15 individual drugs. The three drug combinations listed on the left side of each column are combined with the individual drugs listed at the top of the column for each box painted. The three field combinations described to the right of each column will be combined with the individual drugs listed at the top of the column for each box containing 'X'. This produces a total of 1365 unique combinations of four drugs.

Example 22: Design of a PM test using a tagging system

 The efficiency of the in vitro customized medical examination can be further increased by labeling wells containing different drug compositions with fluorescent probes. The labeled wells can be combined and passed through a flow cytometer, which saves time rather than evaluating each well individually. The achieved savings time may be equal to the number of wells combined. The saved time allows more drug compositions to be tested in less time and allows more testing per ExviTech platform for a unit of time. This leads to an extremely important challenge: increased test volume and reduced costs.

 39 and 40 show examples of multiplexing methods using a fluorescent dye dye. In one embodiment, the fluorescent dye dye is used as a reagent for labeling cells of different drug compositions, for example, contained in different wells. In another embodiment, the fluorescent dye dye is used to label the antibody and then it is used as a reagent for labeling cells in the presence of different drug compositions contained in different wells. In another embodiment, the fluorescent reagent is a quantum dot used to label cells in different drug compositions contained in different wells. In one embodiment, the number of drug compositions that can be evaluated in a multiplexed fashion is in the range defined as about 2, about 5, about 10, about 20, about 30, about 40 or about 50 or any two of the tactical values .

 Figure 39 shows three color multiplexing of peripheral blood leukocytes using different cell tracker dyes. Three consecutive wells containing dissolved peripheral blood were individually stained with different cell tractor dyes. Well 2 was stained with DiR (D12731) (Invitrogen, Carlsbad, Calif.) And Well 3 was stained with DiD (V-22889) (Invitrogen, Carlsbad, Calif.); Well 1 was stained with Pacific Blue (P22652) Carlsbad, Calif.). The contents of the three wells were mixed and obtained at the same time. The unique excitation / emission spectrum of each cell tracker dye allows three distinct cell populations to differentiate into wells of different origin.

Cells from well 1 exhibit stronger signals than cells from wells 2 and 3 in a purple laser detector. Conversely, cells from wells 2 and 3 show a stronger signal in the red laser detector than cells from well 1. Finally, cells in wells 2 and 3 exhibit different emission peaks, which can be separated on a two-dimensional graph of both red laser detectors.

The following references are incorporated herein by reference in their entirety.

1 Leone et al., Cancer Invest 1991, 9: 491-503.

2. Bosanquet et al., Lancet 1991, 337: 711-716.

3. Hongo et al., Cancer 1990; 65: 1263-1272.

4. Hongo et al., Eds. Drug resistance in leukemia and lymphoma. Langhorne, PA: Harwood Academic Publishers 1993, 319.

5. Kaspers et al., Eds. Drug Resistance in Leukemia and Lymphoma. Chur: Harwood Academic Publishers 1993, 321-328.

6. Larsson et al., Int J Cancer 1992, 50: 177-185.

7. Lathan et al., Haematol Blood Transfus 1990, 33: 295-298.

8. Weisenthal LM et al., Cancer Treat Rep 1986, 70: 1283-1295.

9. Weisenthal LM et al., Cancer Res 1983, 43: 749-757.

10. Kirkpatric DL et al., Leuk Res 1990, 14: 459-466.

11. Langker ST et al., Eds. Drug Resistance in Leukemia and Lymphoma. Chur: Harwood Academic Publishers 1993, 279-291.

12. Staib P et al. Adv Exper Med Biol 1999, 457: 437-444.

13. Santini V, et al., Eds. Drug Resistance in Leukemia and Lymphoma. Chur: Harwood Academic Publishers, 1993, 365-368.

14. Sargent JM et al., Br J Cancer 1989, 60: 206-210.

15. Stute N et al., Adv. Exper. Med. Biol 1999, 457: 445-452.

16. Larsson et al., Eds. Drug Resistance in Leukemia and Lymphoma. Chur: Harwood Academic Publishers, 1993: 399-407.

17. Beksac M et al., Med Oncol Tumor Pharmacother 1988, 5: 253-257.

18. Santini V et al., Hematol Oncol 1989, 7: 287-93.

19. Tidefelti et al., Eur J Haematol 1989, 43: 374-384.

20. Bosanquet et al., Br J Haematol 1999, 106: 474-476.

21. Silber et al., Blood 1994, 84: 3440-3446.

22. Bosanquet AG., Eds. Drug Resistance in Leukemia and Lymphoma. Chur: Harwood Academic Publishers 1993, 373-383.

23. Sevin et al., Gynec Oncol 1988, 31: 191-204.

Claims (55)

Obtaining a sample of whole bone marrow taken from a patient having serological neoplasia;
Dividing the whole marrow into at least 35 aliquots, wherein the whole marrow maintains an original cellular microenvironment;
Combining each of said at least 35 fractions with a drug composition; And
And measuring cell depletion in at least one population of cells in each of said at least 35 aliquots. &Lt; Desc / Clms Page number 20 &gt; delete The method according to claim 1,
Wherein the measuring step is completed within 72 hours of combining the fraction with the drug composition. The method according to claim 1,
Wherein the measuring step is completed within 48 hours of combining the fraction with the drug composition. The method according to claim 1,
Wherein the measuring step is completed within 24 hours of combining the fraction with the drug composition. The method according to claim 1,
Characterized in that the number of said aliquots having a unique drug composition is at least 96 or more. The method according to claim 1,
Wherein the sample of the total bone marrow is divided into at least 100 aliquots. The method according to claim 1,
Wherein at least two of the drug compositions comprise the same drug at different concentrations. The method according to claim 1,
Wherein at least one of the drug compositions comprises a plurality of drugs. The method according to claim 1,
Wherein at least one of the drug compositions comprises a plurality of drugs that are non-cytotoxic. The method according to claim 1,
Wherein at least one of the drug compositions comprises a non-cytotoxic drug of the same type or in the same therapeutic region as the drug already administered to the patient. 12. The method of claim 11,
Wherein at least one of the drug compositions is a combination of a non-cytotoxic drug and a cytotoxic drug. The method according to claim 1,
Characterized in that the cell depletion is measured against a population of cells representing the serological neoplasm. 14. The method of claim 13,
The serological neoplasms are: chronic lymphocytic leukemia, adult acute lymphocytic leukemia, childhood acute lymphocytic leukemia, multiple myeloma, myelodysplastic syndrome, non-M3 acute myelogenous leukemia, acute myelogenous leukemia M3, non- Gt; lymphoma, &lt; / RTI &gt; chronic myelogenous leukemia, &lt; RTI ID = 0.0 &gt; gastric &lt; / RTI &gt; lymphoma, Hodgkin's lymphoma, and chronic myelogenous leukemia. 15. The method according to any one of claims 1 and 3 to 14,
Injecting cells obtained from a sample of the whole bone marrow into a mouse;
Allowing the injected cells to proliferate in the mice for a sufficient time; And
Further comprising the step of removing the proliferated cells from the mice, wherein the infusion, proliferation and elimination are performed prior to combining the fractions with the drug composition. The method according to claim 1,
Further comprising the step of generating a report that summarizes the results of said measuring step. 17. The method of claim 16,
Further comprising the step of providing said report to a person concerned with the medical protection of the patient. The method according to claim 1,
The drug composition may be selected from the group consisting of 5-azacytidine, alemtuzumab, aminopterin, amonafide, amsacrine, CAT-8015, bevacizumab, ARR Y520, arsenic trioxide, AS1413, ATRA, AZD 6244, AZD l152, But are not limited to, benzodiazepine, benzoxalantone, bean noirlane-C, vendamustine, bleomycin, binalatum mab, bortezomib, clone, carboplatin, CEP- 701, chlorambucil, chlorodeoxyadenosine, cladribine, Cysteine-linked chain-conjugated anti-CD 19 / cyclophosphamide, cyclophosphamide, cyclosporin, cytarabine, cytosine arabinoside, dasatinib, daunorubicin, decitabine, The compounds of the present invention may be used in combination with anti-CD22 immunotoxins, dexamethasone, doxorubicin, elasitaravin, entinostat, fatu- zum, euinase, etofoside, everonimus, exesecan mesylate, flavopiridol, Dessin, gemcitabine, gemtuzumab - ozogamicin, homoharringtonine, hydrocortisone, ha Iodine I 131 131 monoclonal antibody BC8, isospiritide, isotretinoin, lauromystine, L-asparaginase, lenalidomide, isoleucine, Methylprednisolone, methylprednisone, midosutaurin, mitoxantrone, nelalabine, nilotinib, oblimersen, paclitaxel, panovinostat, pegasaprofen, Paclitaxel, paclitaxel, gabapentin, pentactatin, pyruvicin, PKC412, prednisolone, prednisone, PSC-833, rapamycin, rituximab, ribavirin, sapacitabine, dinacilip, sorafenib, STA-9090, tacrolimus, But are not limited to, spiromycin, temsirolimus, tennyfoside, terameprocole, thalidomide, thioguanine, thiotepa, tififanib, topotecan, thiosulfan, tromsocitabine, vinblastine, Characterized in that it comprises a compound selected from the group consisting of ginsenoside, vinorelbine, borreoxine, borinostat, trivial quidar, and combinations thereof. The method according to claim 1,
Wherein the drug composition is selected from the group consisting of aluminum oxide hydrate, lorazepam, amikacin, melopenem, cefepime, vancomycin, teicoplanin, ondansetron, dexamethasone, amphotericin B (liposomal), caprofugin, itraconazole, fluconazole, A compound selected from the group consisting of acyclovir, voriconazole, trimethoprim, sulfamethoxazole, G-CSF, ranitidine, rasubicase, paracetamol, metamizole, morpholine chloride, omeprazole, paroxetine, fluoxetine, sertraline and combinations thereof &Lt; / RTI &gt; Obtaining a sample of whole bone marrow from a patient who has undergone serological neoplasm diagnosis;
Dividing the whole bone marrow sample into at least 35 aliquots, wherein the whole bone marrow sample maintains an original cellular microenvironment;
When combining each of the at least 35 fractions with a drug composition, the drug composition in each aliquot is different from the drug composition in all other aliquots, at least one of the drug congestion, concentration, or combination thereof, and the drug compositions collectively Comprising at least one non-cytotoxic drug;
Culturing said whole bone marrow sample fraction in combination with a drug composition; And
Comprising analyzing the response of at least one type of neoplastic cell to the drug composition for each cultured product. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; delete 21. The method of claim 20,
Wherein the assay is completed within 72 hours of combining the aliquots with the drug composition. 21. The method of claim 20,
Wherein the assay is completed within 48 hours of combining the aliquots with the drug composition. 21. The method of claim 20,
Wherein the assay is completed within 24 hours of combining the aliquots with the drug composition. 21. The method of claim 20,
Further comprising the step of creating a report that summarizes the results of said analysis step. 26. The method of claim 25,
Further comprising providing said report to a person concerned with the medical care of the patient. 21. The method of claim 20,
Characterized in that the number of aliquots combined with the drug composition is at least 96. 21. The method of claim 20,
RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; wherein said neoplastic cell represents a serological neoplasm. 29. The method of claim 28,
The serological neoplasms are: chronic lymphocytic leukemia, adult acute lymphocytic leukemia, childhood acute lymphocytic leukemia multiple myeloma, myelodysplastic syndrome, non-M3 acute myelogenous leukemia, acute myelogenous leukemia M3, non- Lymphoma, Hodgkin's lymphoma, and chronic myelogenous leukemia. 30. The method according to any one of claims 20 and 22 to 29,
Injecting a mouse with a neoplastic cell obtained from a sample of the whole bone marrow;
Allowing the injected neoplastic cell to proliferate in the mouse for a sufficient time; And
Further comprising the step of removing said proliferating neoplastic cells from said mice, said infusion, proliferation and elimination being performed prior to combining said fractions with a drug composition. 21. The method of claim 20,
The drug composition may be selected from the group consisting of 5-azacytidine, alemtuzumab, aminopterin, amonafide, amsacrine, CAT-8015, bevacizumab, ARR Y520, arsenic trioxide, AS1413, ATRA, AZD 6244, AZD 152, But are not limited to, benzodiazepine, benzoxalantone, bean noirlane-C, vendamustine, bleomycin, binalatum mab, bortezomib, clone, carboplatin, CEP- 701, chlorambucil, chlorodeoxyadenosine, cladribine, Cysteine-linked chain-conjugated anti-CD 19 / cyclophosphamide, cyclophosphamide, cyclosporin, cytarabine, cytosine arabinoside, dasatinib, daunorubicin, decitabine, The compounds of the present invention may be used in combination with anti-CD22 immunotoxins, dexamethasone, doxorubicin, elasitaravin, entinostat, fatu- zum, euinase, etofoside, everonimus, exesecan mesylate, flavopiridol, Dessin, gemcitabine, gemtuzumab - ozogamicin, homoharringtonine, hydrocortisone, ha Iodine 1 131 monoclonal antibody BC8, Isofemide, isotretinoin, lauromystine, L-asparaginase, lenalidomide, lidocaine, lidocaine, Methylprednisolone, methylprednisone, midosutaurin, mitoxantrone, nelalabine, nilotinib, oblimersen, paclitaxel, panovinostat, pegasaprofen, Paclitaxel, paclitaxel, gabapentin, pentactatin, pyruvicin, PKC412, prednisolone, prednisone, PSC-833, rapamycin, rituximab, ribavirin, sapacitabine, dinacilip, sorafenib, STA-9090, tacrolimus, But are not limited to, spiromycin, temsirolimus, tennyfoside, terameprocole, thalidomide, thioguanine, thiotepa, tififanib, topotecan, thiosulfan, tromsocitabine, vinblastine, Characterized in that it comprises a compound selected from the group consisting of ginsenoside, vinorelbine, borreoxine, borinostat, trivial quidar, and combinations thereof. 21. The method of claim 20,
Wherein the drug composition is selected from the group consisting of aluminum oxide hydrate, lorazepam, amikacin, melopenem, cefepime, vancomycin, teicoplanin, ondansetron, dexamethasone, amphotericin B (liposomal), caprofugin, itraconazole, fluconazole, Selected from the group consisting of voriconazole, trimethoprim, sulfamethoxazole, G-CSF, ranitidine, rasburicase, paracetamol, metamizole, morpholine chloride, omeprazole, paroxetine, fluoxetine, sertraline, and combinations thereof Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; Providing a whole bone marrow sample comprising neoplastic cells obtained from a patient;
Culturing each of at least six portions of the whole bone marrow sample with a different drug or drug combination, wherein the whole marrow maintains an original cellular microenvironment;
Analyzing each of the portions of the bone marrow sample to determine the extent of cell depletion of neoplastic cells in the portion; And
Characterized in that the method comprises the step of issuing a printed or electronic report of the results from the analysis step, indicative of at least the portion, drug or drug combination having a maximum degree of cell depletion. A method of facilitating the treatment of a neoplasm. 34. The method of claim 33,
Wherein the resulting report is indicative of results from a plurality of drugs or combinations of drugs. A plurality of chambers; An apparatus for analyzing the response of a neoplastic cell from a sample of whole bone marrow to a potential drug regimen comprising a different drug or drug combination in each of the plurality of chambers, Wherein said whole bone marrow maintains a round cell microenvironment, said chambers collectively comprising:
At least one chamber comprising a plurality of medicaments;
At least one chamber comprising a cytotoxic drug;
At least one chamber comprising a non-cytotoxic drug; And
Characterized in that it comprises at least a total of 10 different drugs in a collective chamber. 36. The method of claim 35,
Wherein the at least one chamber comprises a cytotoxic drug and a non-cytotoxic drug. 36. The method of claim 35,
Characterized in that it further comprises at least two chambers containing the same drug at different concentrations. 36. The method of claim 35,
Wherein the at least one chamber comprises fluaravarin or chlorambucil in combination with sertraline, paroxetine, or fluoxetine. 36. The method of claim 35,
Wherein the at least one chamber comprises fluvastatin and cyclophosphamide. 40. The method according to any one of claims 35 to 39,
At least one of the at least 10 different drug compositions may be selected from the group consisting of 5-azacytidine, alemtuzumab, aminopterin, amonafide, amsacrine, CAT-8015, bevacizumab, ARR Y520, arsenic trioxide, CEP-701, chloramphenicol, chlorobenzene, chloramphenicol, chloramphenicol, chloramphenicol, chloramphenicol, chloramphenicol, chloramphenicol, Cyclophosphamide, cyclosporin, cytarabine, cytosine arabinoside, dasatinib, daunorubicin, decitabine, deglycosylated-lysine-A Chain-conjugated anti-CD 19 / anti-CD22 immunotoxins, dexamethasone, doxorubicin, elacitarabine, entinostat, efrafuzumab, euinase, etoposide, everonimus, exatexan mesylate, flavor Pyridol, Fludarabine, Porodecin, Gemcitabine, Gemtuzumab - Ozogamicin, Ho Isoindine 1 131 monoclonal antibody BC8, ifosfamide, isotretinoin, ra- romustine, L-asparaginase, interferon alpha 2a, iodine 1 131 monoclonal antibody BC8, But are not limited to, nausea, lenalidomide, lestaurin tinib, mapospamide, melphalan, mercapturine, methotrexate, methylprednisolone, methylprednisone, midosutaurin, mitoxantrone, nelalabine, neilotinib, oblimersen, paclitaxel, Pantovastatin, Pyrarubicin, PKC412, Prednisolone, Prednisone, PSC-833, Rapamycin, Rituximab, Ribavirin, Sapacitabine, Dinacilip, Sorapenib, STA- 9090, Tacrolimus, Tanepsimycin, Temsirolimus, TenniPoside, Terrameproc, Thalidomide, Thioguanine, Thiotepa, Tifipanib, Topotecan, Thiosulfan, Blank, vinblastine, vincristine, binde new, unexposed mozzarella bar, Borel roksin, barley furnace stats, assistant quinolyl different, and wherein is selected from the group consisting of a combination thereof, the device. 40. The method according to any one of claims 35 to 39,
At least one of the total of at least 10 different drug compositions is selected from the group consisting of aluminum oxide hydrate, lorazepam, amikacin, melopenem, cephapyr, vancomycin, teicoplanin, ondansetron, dexamethasone, amphotericin B (liposomal) But are not limited to, carfosfugin, itraconazole, fluconazole, voriconazole, trimethoprim, sulfamethoxazole, G-CSF, ranitidine, rasburicac, paracetamol, metamizole, morphine chloride, omeprazole, paroxetine, fluoxetine, sertraline, &Lt; / RTI &gt; and combinations thereof. 40. The method according to any one of claims 35 to 39,
Wherein the neoplastic cell represents multiple myeloma (MM), and at least one of the chambers comprises: rubicin plus cytarabine + VP-16, daunorubicin plus cytarabine, dirubicin plus cytarabine, , At least one drug combination selected from the group consisting of mitoxantrone + cytarabine + VP-16, atra + dirubicin and cytarabine + mitoxantrone + atra. 40. The method according to any one of claims 35 to 39,
Wherein the at least one of the chambers is selected from the group consisting of cyclophosphamide + doxorubicin + vincristine + prednisolone, cyclophosphamide + doxorubicin + prednisolone, fludarabine + cyclophosphorus Id + rituximab, pentostatin + cyclophosphamide + rituximab, fludaravine + cyclophosphamide + oppartum, pentostatin + cyclophosphamide + oppartum, fluaravarin + cyclophosphamide At least one drug combination selected from the group consisting of id + alemtuzumab and pentostatin + cyclophosphamide + alemtuzumab. 40. The method according to any one of claims 35 to 39,
Wherein the neoplastic cell exhibits acute lymphocytic leukemia (ALL) and at least one of the chambers is selected from the group consisting of vincristine + daunorubicin + prednisona, vincristine + prednisone + mitoxantrone + cytarabine, methotrexate + Lavin + hydrocortisone, dexamethasone + vincristine + methotrexate + cytarabine + L-asparaginase + 6-mercaptopurine, cyclophosphamide + doxorubicin + vincristine + dexamethasone, dexamethasone + daunorubicin + cyclophos Methamphetamine + L-asparaginase, vincristine + prednisone, methotrexate + ethofoside + cytarabine + thioguanine, methotrexate + 6-mercaptopurina, vincristine + daunorubicin + Phosphamide + Prednisona, TenniPoside + Cytarabine, Vincristine + Daunorubicin + Cyclophosphamide + L - Asparaginase + dexamethasone, vincristine + L-asparaginase, vincristine + daunorubicin + cytarabine + L- asparaginase + imatinib + prednisone, mitoxantrone + cytarabine + imatinib, methotrexate + imatinib + 6 Which comprises at least one drug combination selected from the group consisting of mercaptopurine, teniposide + cytarabine + imatinib and vincristine + daunorubicin + cyclophosphamide + L-asparaginase + dexamethasone + / RTI &gt; 39. The method according to any one of claims 35 to 39,
Wherein said neoplastic cell represents a non-Hodgkin's lymphoma (NHL), at least one of said chambers being selected from the group consisting of cyclophosphamide + doxorubicin + vincristine + prednisone, cyclophosphamide + doxorubicin + vincristine + prednisone + rituximab , Cyclophosphamide + doxorubicin + vindecina + prednisone, cyclophosphamide + doxorubicin + vindesina + prednisone + interferon alpha, cyclophosphamide + vincristine + prednisone, cyclophosphamide + vincristine + prednisone + rituxi Methotrexate, doxorubicin + vincisin + prednisone + bromomycin, methotrexate, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, + Etoposide + ifosfamide + cytarabine, methotrexate + binx Cyclophosphamide + Doxorubicin + Cyclophosphamide + Prednisone +, Vincristine + Bleomycin + Prednisone +, Dexamethasone + Citaravine + Cisplatin +, Fluaravine + Cyclophosphamide + Mitoxantrone, Cyclophosphamide + Doxorubicin + Vincristine + dexamethasone, methotrexate + hydrocortisone + cytarabine + dexamethasone + cyclophosphamide, vendamustine + mitoxantrone, ifosfamide + carboplatin + etoposide + rituximab and etoposide + prednisone + bin Wherein the drug combination comprises at least one drug combination selected from the group consisting of Christine + cyclophosphamide + doxorubicin + rituximab. 40. The method according to any one of claims 35 to 39,
Wherein said neoplastic cell exhibits acute myeloid clots (AML), and at least one of said chambers comprises rubicin plus cytarabine + VP-16, daunorubicin plus cytarabine, dirubicin plus cytarabine, Ribavirin + mitoxantrone + atra, daunorubicin + cytarabine + thioguanine, daunorubicin + cytarabine + VP-16 G-CSF, fluarazine + cytarabine + G-CSF, high dose cytarabine + VP- 16 + daunorubicin, gemtuzumab ozogamicin + rubicin + VP-16, mitoxantrone + VP-16, dirubicin, clindamycin + citalin + cloparazine + cytarabine + dirubicin, amsacrine + cytarabine + VP-16, mitoxantrone + + Cytarabine + FLT3 inhibitor, cytarabine + FLT3 inhibitor, cytarabine + aurora kinase Gatifoumine, cladribine + dirubicin + cytarabine, decitabine + valproic acid, dicitabine + valproic acid, fludarabine + isarubicin + cytarabine + Jenna Sense + Flu Da Rabin + Cytarabine, Jenna Sense + Daunorubicin + Cytarabine, Jenna Sense + Cytarabine, Jenna Sense + Gentoo Zuma The Ojo Glimmy, PSC833 + Daunorubicin + Cytarabine, PSC833 + Characterized in that it comprises at least one drug combination selected from the group consisting of + cytarabine, PSC833 + daunorubicin + cytarabine + VP-16 and bortezomib + rubicin + cytarabine. delete delete delete delete delete delete delete delete delete

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