Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Non-toxic amounts of protein-aggregating substances stimulate Hsp70 expression and function as anti-tumor agents
• the present invention relates to the use of a drug inducing intracellular protein aggregation for the preparation of a pharmaceutical composition wherein the drug is in a dose of 135-175 mg/m 2 body surface for the treatment of a tumor, a bacterial infection or a viral infection.
• the drug is selected from vincristine and paclitaxel.
• the present invention further relates to a method of treating a patient suffering from a tumor, a bacterial infection or a viral infection comprising administering to said patient a drug inducing intracellular protein aggregation in a dose of 135-175 mg/m 2 body surface. It is preferred that said drug is administered in combination with heat treatment.
• HSP heat shock proteins
• HSP-peptide complexes into antigen presenting cells APC
• HSP-chaperoned tumor-derived peptides are re-presented on major histocompatibility antigen complex (MHC) class I complexes into antigen presenting cells (APC).
• MHC major histocompatibility antigen complex
• APC antigen presenting cells
• HSP-chaperoned tumor-derived peptides are re-presented on major histocompatibility antigen complex (MHC) class I molecules, and thus elicit a CD8 mediated T cell immune response (Basu et al.
• Hsp70 high cytoplasmic Hsp70 levels have been reported to contribute to anti- apoptotic mechanisms in different tumor cell systems (Wei et al. 1994; Gabai et al. 1998; Jaattela et al. 1998). Because of this dual activity of Hsp70, it is important to study not only the amount, but also the cellular and subcellular localization of Hsp70, to understand its specific immune function. High intracellular Hsp70 levels in prostate carcinoma cells are associated with drug resistance (Roigas et al. 1998).
• Cytarabine and ifosfamide are potent antineoplastic drugs with a broad spectrum of biological activities. Both compounds affect replicating cells and interact with DNA. Cytarabine, a pyrimidine antagonist that is converted in arabinosyl-cytosintriphosphat by kinases, is most efficient in the S phase of the cell cycle and therefore predominantly affects viability of rapidly growing cells (Grant 1998). Cytarabine undergoes phosphorylation by deoxycytidine kinases before its incorporation into DNA, which finally results in cell death. Clinically, cytarabine is applied in the treatment of acute lymphoblastic leukemia (ALL), Non-Hodgkin lymphoma (NHL) and acute myelogenous leukemia (AML).
• ALL acute lymphoblastic leukemia
• NHL Non-Hodgkin lymphoma
• AML acute myelogenous leukemia
• ifosfamide is metabolically activated by hepatic mixed-function oxidases into 4-hydroxyifosf amide (4-OH-IF) which is decomposed to alkylating mustard (Zalupski and Baker 1988; Multhoff et al. 1995c).
• 4-hydroxyifosf amide (4-OH-IF) which is decomposed to alkylating mustard (Zalupski and Baker 1988; Multhoff et al. 1995c).
• the DNA-alkylating and crosslinking activity of the mustard induces cell death especially in proliferating cells.
• the remaining metabolites are acrolein, aldocyclophosphamide/ aldoifosfamide and chloroacetaldehyde which are produced alternatively by beta-oxidation form conjugates with glutathione.
• ifosfamide In order to mimick the metabolically active form of ifosfamide, for in vitro investigations the prodrug 4- hydroperoxyifosfamide (4-OOH-IF) was used, which in aqueous solutions rapidly gives rise to pharmacologically equivalent amounts of the activated form of ifosfamide (4-OH-IF).
• Vincristine is used either as a single agent or in combination with ifosfamide, methotrexate, and cytarabine in a number of solid tumors and in hematological malignancies including ALL, Acute Non-Lymphoblastic Leukemia (ANLL) or Non-Hodgkin Lymphoma (NHL).
• Vincristine is known to bind microtubular proteins, inhibits the mitotic spindle formation and thus causes an arrest in the metaphase of mitosis (Gidding et al. 1999).
• Paclitaxel the effective compound of Taxol-100 is used for the treatment of ovarian and mammary carcinomas (Eisenhauer et al. 1998). It is assumed that paclitaxel binds to tubulin dimers and thus disables the dynamic reorganization of the tubular network during the active interphase and during mitosis. It supports the formation of microtubuli aggregates and inhibits their depolymerization (Kingston 2000; Snyder et al. 2001).
• a significant disadvantage of the state of the art cancer treatment using any of the above recited anti-tumor drugs is the recognized necessity to administer said drugs in relatively high concentrations. These high concentrations are in vivo toxic not only for tumor cells but also for non-tumorous cells. As a consequence, a patient treated with the prescribed regimen of any of those drugs suffers from severe adverse effects. Similar problems with severe side effects are often encountered in the treatment of bacterial or viral infections.
• the technical problem underlying the present invention was to provide means and methods for an essentially efficacious treatment, in particular of tumors but also of bacterial or viral infections that is not accompanied by said severe adverse effects.
• the present invention relates to the use of a drug inducing intracellular protein aggregation for the preparation of a pharmaceutical composition wherein the drug is in a dose of 135-175 mg/m 2 body surface for the treatment of a tumor, a bacterial or viral infection.
• intracellular protein aggregates is intended to mean an aggregate of proteins that does not normally or under physiological conditions occur in a viable, non-tumorous cell.
• Hsp70 is induced by vincristine (V) and paclitaxel (P) in the cytoplasma of normal cells (a corresponding experiment is shown in Fig 2C).
• Such aggregates may consists of the same as well as of different proteins.
• protein aggregates may be formed upon the application of stress, e.g. in the form of heat, radiation or chemicals, to a cell. The formation of said aggregate eventually renders the cell bound to cell death.
• Hsp70 is induced in healthy cells, i.e. PBL (a corresponding experiment is shown in Fig 4B).
• the teaching of the present invention therefore provide a much improved approach to the treatment of a large variety of tumors or of bacterial or viral infections without the severe adverse effects that were concomitantly observed with the prior art drug anti-cancer etc. treatment. It is to be noted that the concentration range of the drugs is in accordance with the present invention, clearly a range sublethal for normal cells.
• the present invention may also be viewed as the use of drug inducing exposure of Hsp70 on the surface of tumor or infected cells for the preparation of a pharmaceutical composition wherein the drug is in a dose of 135 - 175 mg/m 2 body surface for the treatment of a tumor or of bacterially or virally infected cells.
• Viruses include HIV such as HIV I and HIV II as well as adenovirus. Further viruses include EBV (Epstein-Barr Virus), CMV (Cytomegalivirus), Coxsachievirus, Herpes simplex virus, HPV (human papilloma virus) and Human T-cell leukemia virus.
• Bacteria include Mycobacterium tuberculosis and Listeria.
• protein aggregation in cells may be assessed by means of an anti- tubulin antibody that is fluorescently labeled. Aggregates of tubulin are detected as clumps on the basis of the fluorescence intensity and using, for example, a fluorescence microscope.
• the occurrence of Hsp70 on the cell surface of tumor cells may be measured using the technology described in the appended examples.
• One convenient method to assess Hsp70 occurrence as density on the surface of tumor cells or infected cells is flow cytometry.
• inducing intracellular protein aggregation is intended to mean that the drug, upon administration, is causative for the onset or occurrence of protein aggregation.
• the drug may directly induce aggregation by acting on the proteins that form the aggregate.
• the drug may have an indirect effect on the onset or occurrence of protein aggregation.
• the drug may interact with a protein in a signal cascade wherein the signal cascade eventually leads to protein aggregation.
• compositions prepared in accordance with the present invention may further comprise a pharmaceutically acceptable carrier and/or diluent.
• suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
• Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration.
• compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
• Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
• the term "wherein the drug is in a dose of 135-175 mg/m 2 body surface” describes the dosage of active ingredient required in accordance with the present invention. It is important to note that this dosage regimen is non-toxic for non-tumorous or non- infected cells. This dose corresponds to a serum content of 10 vM or lower. The person skilled in the art is easily in a position to transform this value into a different commonly used prescription of the dose, e.g. in a dose calculated based on the amount of drug (in milligrams) to be administered per kilogram of body weight.
• the claimed invention specifically includes embodiments wherein the prescribed dosage is administered is administered more than once, such as two, there, four, five, six, seven, eight, nine, ten or more times.
• one dose is already effective in the induction of surface exposition of Hsp70 by tumor cells or infected cells. Accordingly, a clinical effect is already observed after a single dose treatment. Administration of multiple doses will, as a rule, enhance the clinical effect.
• tumor includes malignant as well as non-malignant tumors.
• a malignant tumor differs from a non-malignant tumor by its ability to invade surrounding tissues.
• the drug is an anti-cancer drug, i.e. a drug that was known in the art to selectively kill tumor cells and preferably malignant tumor cells.
• said drug binds to microtubuli and interferes with the progress of mitosis. Accordingly, it is also preferred that said drug induces the formation of tubulin aggregates.
• the present invention demonstrates the capacity of antitumor agents that induce protein aggregation in cells to induce Hsp70, the major heat-inducible member of the 70kDa heat shock protein family. From the prior art, it was known that on the one hand, elevated levels of Hsp70 are discussed to play a crucial role in the induction of anti-apoptotic mechanisms that enable tumor cells to escape from apoptotic cell death induced by stimuli including heat shock, tumor necrosis factor, oxidative stress, cytostatic drugs, and radiation (Roigas et al.
• Hsp70 extracellular Hsp70 induces NK activity (Multhoff et al. 1999) and plasma membrane-bound Hsp70 has been determined as a tumor-selective target recognition structure for NK cells (Multhoff et al. 1997).
• Hsp70 was not only found on the cell surface of tumor cell lines but also on freshly isolated biopsy material of carcinoma and leukemic patients (Hantschel et al. 2000).
• K562 cells ATCC CCL 243
• nontoxic doses of drugs inducing intracellular protein aggregation such as vincristine or paclitaxel not only induce increased cytoplasmic but also membrane-bound Hsp70 levels.
• the antitumor agents that interact with DNA neither increase the amount of cytoplasmic nor enhance the amount of membrane-bound Hsp70 in tumor cells (see appended examples). This is in agreement with the idea of Hightower that Hsp70 induction is a sensitive marker for proteotoxicity rather than genotoxicity (Hightower 1991).
• Hsp70 membrane localization was restricted to tumor cells or infected cells; PBL derived from healthy human individuals did not exhibit any cell surface localization either under physiological conditions or following stress (Multhoff et al. 1995a, 1995b).
• treatment with heat alone or with heat plus tubulin interacting agents also did not increase membrane-bound Hsp70 on PBL.
• Hsp70 membrane localization induced by vincristine or paclitaxel is tumor cell specific or specific for cells infected by bacteria or viruses.
• the immunological consequences of increased amounts of membrane-bound Hsp70 were shown for various tumors including human colon carcinoma cells and K562 cells.
• the sensitivity to lysis mediated by NK cells was drastically increased following incubation with the above-mentioned drug type and in particular with a nontoxic dose of paclitaxel.
• the findings of the present invention are particularly advantageous since they allow the treatment of patients suffering from tumors or bacterial or viral infections with doses of medicaments that are clearly below the level of toxicity required for the killing of non-tumorous or non-infected ceils.
• the non-toxic value for vincristine is between 0 and 250 ⁇ M when tested in vitro with K562 cells; see Figure 6 and Table 2.
• the value for paclitaxel is between 0 and 10 ⁇ M, see Figure 7 and Table 2.
• the range in which the drugs stimulate Hsp70 surface expression is marked by bars.
• the phenomenon of intracellular protein aggregation could be linked with the fact that tumor cells are susceptible to the pharmaceological effects of anti-cancer drugs administered at a much lower dose than prescribed in state of the art anti-cancer pharmaceutical compositions.
• the person skilled in the art may recur to further anti-cancer drugs as drugs effective in the treatment of viral or bacterial infections having the same effects on intracellular protein aggregation and not exemplified in this specification with the sound expectation that these drugs efficaciously eradicate tumor or infected cells when administered at nontoxic levels.
• the dosages of the drug deviate from the range indicated above but are nevertheless in a non-toxic range.
• Another advantage of the present invention is that many of the drugs that fall under the scope of this invention have been approved and registered for essentially the same indications that are followed up here.
• the use of said drug in the preparation of a pharmaceutical composition for he treatment of a tumor or a bacterial or viral infection wherein the drug yields plasma levels after administration of 5 to 10 ⁇ M and induces intracellular protein aggregation or cell surface expression of Hsp70 is another preferred embodiment of the invention.
• the administration of the drug in the above indicated range will yield plasma levels between 5 and 10 ⁇ M.
• the drugs can be tested in appropriate systems in ranges essentially between 1 nM and 300 ⁇ M wherein levels of 10 nM to 10 ⁇ M (for paclitaxel) or 10nM to 250 /M (for vincristine) indicate pharmacological activity as well as non-toxicity of the drugs.
• said antineoplastic drug is vincristine.
• Vincristine targets ⁇ -tubulin and thus results in tubulin assembly.
• Vincristine supports the assembly of tubulin and thus causes protein aggregation within the cell (Gidding et al. 1999);
• the drug administered is Vincristin Liquid, the clinically applied homologue of vincristine.
• Vincristine Liquid is manufactured and distributed by Eli Lilly.
• said drug is paclitaxel.
• paclitaxel targets ⁇ -tubulin and thus induces tubulin assembly. Further, paclitaxel blocks mitosis by binding and stabilizing microtubules (Schiff and Horwitz 1980).
• said drug is Taxol - 100, the clinically applied homologue of paclitaxel.
• Taxol ® -100 is manufactured and distributed by Bristol Myers Squibb.
• the tumor treated in accordance with the present invention may be a benign tumor.
• said tumor is a malign tumor.
• the malign tumor may either be a solid tumor or a hematological malignancy.
• said malign tumor is a tumor selected from the following: Colorectal, pancreas, melanoma, lung carcinoma (small and non-small), ovarian, kidney, mammary, head and neck, stomach, liver, oesophagus, prostata carcinomas, and sarcomas.
• said hematological malignancy is selected from myeloproliferative diseases, non-Hodgkin's Iymphoma (NHL), AML (acute myelocytic leukemia), acute lymphocytic leukemia (ALL), multiple myeloma, chronic myelocytic leukemia (CML) and chronic lymphocytic leukemia (CLL), B- and T-lymphomas, CNS lymphomas, gastrointestinal lymphomas, and cutaneous lymphomas.
• myeloproliferative diseases non-Hodgkin's Iymphoma (NHL), AML (acute myelocytic leukemia), acute lymphocytic leukemia (ALL), multiple myeloma, chronic myelocytic leukemia (CML) and chronic lymphocytic leukemia (CLL), B- and T-lymphomas, CNS lymphomas, gastrointestinal lymphomas, and cutaneous lymphomas.
• the present invention furthermore relates to a method of treating a patient suffering from a tumor or a bacterial or viral infection comprising administering to said patient a drug inducing intracellular protein aggregation in a dose of 135-175mg/m 2 body surface.
• said drug is vincristine.
• said drug is paclitaxel.
• said tumor is a malign tumor.
• said malign tumor is a tumor selected from the following: Colorectal, pancreas, melanoma, lung carcinoma (small and non-small), ovarian, kidney, mammary, head and neck, stomach, liver, oesophagus, prostata carcinomas, and sarcomas.
• said hematological malignancy is selected from myeloproliferative diseases, non-Hodgkin's lymphoma (NHL), AML (acute myelocytic leukemia), acute lymphocytic leukemia (ALL), multiple myeloma, chronic myelocytic leukemia (CML) and chronic lymphocytic leukemia (CLL), B- and T-lymphomas, CNS lymphomas, gastrointestinal lymphomas, and cutaneous lymphomas.
• myeloproliferative diseases non-Hodgkin's lymphoma (NHL), AML (acute myelocytic leukemia), acute lymphocytic leukemia (ALL), multiple myeloma, chronic myelocytic leukemia (CML) and chronic lymphocytic leukemia (CLL), B- and T-lymphomas, CNS lymphomas, gastrointestinal lymphomas, and cutaneous lymphomas.
• the method further comprises the administration of heat in the range between > 37°C to 43°C for 1 to 12 hours to said patient.
• heat is also administered in a sublethal dose, also when administered in conjunction with the drug.
• This embodiment of the present invention is particularly advantageous due to the fact that an additive effect could be observed when heat was administered in addition to the drug.
• the heat may be administered simultaneously, or it may be administered prior to administration of the drug. However, care needs to be taken that there is a time overlap of the pharmacological effect due to the application of the drug and the heat treatment. Generally, if the patient is treated at a higher temperature, the time of heat administration may be shorter than if the patent is treated at a low temperature. It must be emphasized, however, that the general range of the drug goes well with the indicated heat range. A preferred range is 38°C to 41 °C.
• Heat may be administered to a certain part of the patient's body or a whole body by hyper thermal treatment. Sources of heat includes microwaves (companies distributing appropriate instruments include BSD, Sennewald, Sigma Eye, Siemens) infrared radiation, and hot-air.
• the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a drug inducing intracellular protein aggregation in a dose of 135-175 mg/m 2 body surface.
• the drug interacts with tubulin, as has been described above.
• the drug is vincristine or paclitaxel (particularly Vincristin Liquid or Taxol ® -100).
• the pharmaceutical composition may comprise one or more doses of the drug to be formulated and administered as described above.
• K562 cells were incubated with Camptothecin (Campto) at a concentration of 2 ⁇ g/ ml.
• cytoplasmic Hsp70 The relative increase of cytoplasmic Hsp70 is indicated for each antineoplastic agent graphically and as numbers (fold increase) on the right hand side.
• Cytoplasmic Hsp70 levels were increased in PBL of healthy donors following treatment with vincristine (V) or paclitaxel (P) with or without heat.
• Fig 4. A) Flow cytometric analysis of membrane-bound Hsp70 on K562 tumor cells.
• Untreated (37°C) or heat-shocked (41.8°C) K562 cells were incubated with 1 ⁇ M of the cytostatic drugs vincristine (V) or paclitaxel (P) and stained with a FITC- conjugated Hsp70 specific antibody (C92 F3 B1) (solid line) or isotype-matched controls (dotted line). The percentage of Hsp70 positively stained cells is shown in the right corner of each graph.
• Hsp70 cell surface expression was not induced in PBL of healthy human donors following vincristine (V) or paclitaxel (P) treatment.
• Untreated (37°C) or heat-shocked (41.8°C) PBL derived from healthy donors (n 5) were treated either with 1 ⁇ M V or 1 ⁇ M P and stained with FITC-conjugated Hsp70 antibody C92FB1.
• the marker indicates the position of the isotype-matched (lgG1) control antibody.
• the % Hsp70 positively stained cells is shown in the upper right corner of each graph.
• Untreated (37°C) or heat-shocked (41.8°C) K562 cells were incubated with 1 ⁇ M of the cytostatic drugs vincristine (V) or paclitaxel (P). Either untreated (ctrl) or vincristine (V) and paclitaxel (P) treated cells were mounted on glass slides, fixed with 4% paraformaldehyd, incubated with a tubulin specific monoclonal antibody and stained with a secondary FITC-conjugated antibody. Scale bars; 10 ⁇ m.
• Peripheral blood lymphocytes (PBL) were derived from heparinized blood of healthy human volunteers following separation by Ficoll density gradient centrifugation. After washing the cells were counted and resuspended in the same medium that was used for K562 cells.
• Example 1 Treatment with cytostatic drugs
• 4-OH-IF is decomposed to alkylating mustard, when acrolein is split off.
• aqueous solution of 4-OOH-IF was freshly prepared directly before each assay (Zalupski and Baker 1988; Multhoff et al. 1995b).
• the DNA intercalating cytostatic drugs carboplatin (Ribocarbo-L, ribosepharm, M ⁇ nchen, Germany), doxorubicin (Doxo-cell, cell pharm, Hannover, Germany), fludarabine (Fludara, Schering, Berlin, Germany), cytarabine (Ara-cell, cell pharm, Hannover, Germany), and the tubulin interacting agents vincristine (Vincristin Liquid, Lilly, Giessen, Germany), and paclitaxel (Taxol-100, Bristol, Kunststoff, Germany), were obtained from the pharmaceutical department of the University Hospital Regensburg. All agents were prepared freshly as described for medical applications.
• cytoplasmic fractions were prepared from 5 x 10 6 cells by incubation of PBS washed cell pellets in 10 mM Tris-buffered saline (pH 7.5) containing 1% Nonidet P-40 (NP-40; Sigma) and 1 mM phenyl-methyl-sulphonyl- fluoride (PMSF; Sigma) as described previously (Botzler et al. 1999).
• Plasma membrane fractions were prepared by a modified method described by Weissman (1991). Briefly, 50 x 10 6 cells were broken by 30 strokes with a tight pestle in a Dounce homogenizer.
• the cytoplasmic fraction was separated from the membrane fraction by ultracentrifugation at 100,000 g at 4°C.
• Membrane-bound proteins were separated by treatment of the last pellet with Triton X-100 (Sigma) followed by centrifugation of the unsoluble material at 10,000 g.
• the myelogenous tumor cell line K562 (CCL243, ATCC) and the human colon carcinoma cell line CX2 (see below; TZB 61005, Tumorbank DKFZ, Heidelberg, Germany) (Multhoff et al. 1997) were grown in RPMI 1640 medium (GibcoBRL, Eggenstein, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS, BioWhittaker, Walkersville, Maryland, USA), 6 mM L-glutamine, and antibiotics (100IU/ ml penicillin and 100 ⁇ g/ ml streptomycin, GibcoBRL, Eggenstein, Germany). In order to obtain exponential growth the cell density was maintained at 0.5 x 10 6 .
• a concentration of 10O ⁇ M of the antineoplastic drugs negatively affects the viability of the cells.
• the reduction in cell viability at 100 ⁇ M was less pronounced if the cells were treated with a nonlethal heat shock (41.8°C) for 2 hours before the incubation with the cytostatic drugs. (Briefly, exponentially growing tumor and normal cells were treated at a nonlethal temperature of 41.8°C for 2 hours in a temperature-controlled waterbath (GFL, Burgwedel, Germany) followed by a recovery period of 15 hours at 37°C. Under these conditions the cell viability was greater 99%.)
• the concentrations, 10nM, 1 ⁇ M, and 10 ⁇ M of the antineoplastic drugs, either under physiological conditions or combined with heat, have been determined as nontoxic for K562 cells.
• annexin staining Briefly, cells were washed twice in Hepes buffer containing 5 mM CaCI 2 and incubated with Annexin-V-FLUOS (Roche) for 10 min at room temperature. Annexin positively stained cells were measured in a FACSCalibur flow cytometer. At a concentration of 1 ⁇ M none of the drugs induces significant apoptosis in either untreated (37°C) or heat-shocked (41.8°C) K562 cells 24 hours following treatment.
• Hsp70 in K562 cells maintained under physiological conditions (37°C) or following nonlethal heat shock (41.8°C), were treated with cytarabine (C), the activated form of ifosfamide (I), vincristine (V), or paclitaxel (P) at the nontoxic concentrations 10nM, 1 ⁇ M, 10 ⁇ M and at the lethal concentration of 100 ⁇ M.
• C cytarabine
• I the activated form of ifosfamide
• V vincristine
• P paclitaxel
• the colon carcinoma cells CX2 also was treated with the different cytostatic drugs at the nonlethal concentration of 1 ⁇ M. Similar to K562 cells, cytarabine (C) and ifosfamide (I) did not influence the cytoplasmic amount of Hsp70, whereas, vincristine (V) and paclitaxel (P), both increase the cytoplamic amount of Hsp70 in the range of 20 to 25%. Furthermore, the question as to whether a combined treatment consisting of nonlethal heat plus antineoplastic agents exhibits a synergistic effect on the cytoplasmic Hsp70 levels was posed. In line with previously published data (Botzler et al.
• Example 5 Effects of antineoplastic agents on the amount of membrane-bound Hsp70 on tumor cells Beside its chaperoning function, extracellular localized Hsp70 is known to stimulate NK cell activity.
• Cell membrane-bound Hsp70 acts as a tumor-selective target recognition structure (Multhoff et al. 1995a; Multhoff et al. 1995c).
• C cytarabine
• I ifosfamide
• V vincristine
• P paclitaxel
• Hsp70 membrane expression could be confirmed by flow cytometric analysis. Briefly, indirect immunofluorescence studies were performed using the Hsp70 specific monoclonal antibody (MoAb, clone C92 F3 B1 , multimmune GmbH, Regensburg, Germany), the MHC class I specific MoAb (W6/32) and isotype- matched control antibodies (lgG1 , lgG2a Immunotech, Marseille, France) as primary antibodies and FITC- or PE-conjugated rabbit anti-mouse secondary antibodies (Dako, Hamburg, Germany). Briefly, the cells were incubated with the primary antibodies at 4°C for 30 min. After two washing steps the cells were stained with a secondary antibody for another 30 min at 4°C.
• a quantitative flow cytometric analysis was performed using a FACSCalibur instrument (Becton Dickinson, Heidelberg, Germany). The percentage of positively stained cells was determined as the number of positively stained cells minus the number of cells stained with an isotype-matched negative control antibody. Only viable, propidium iodide (PI) negative cells were analysed.
• PI propidium iodide
• Example 6 Effects of antineoplastic agents vincristine (V) and paclitaxel (P) on the formation of tubulin aggregates in tumor cells
• tumor cells were treated with 1 ⁇ M vincristine (V) or paclitaxel (P) for 2 hours. Following a recovery period of 2 hours at 37°C the cells were washed and settled on poiy-L-lysine coated glass slides, fixed with 4% paraformaldehyde in PBS for another 30 min at room temperature. After fixation the cells were incubated with an antibody directed against tubulin (AB-1 , Oncogene, Boston, USA) and stained with a goat-anti-mouse FITC conjugated secondary antibody (Dako, Glostrup, Denmark) for another 30 min.
• V vincristine
• P paclitaxel
• the slides were mounted with Fluorescent Mounting Medium (Dako, Carpinteria, USA) and then the samples were analysed for transmission and fluorescence using a Zeiss model Axioscop 2 scanning microscope (Zeiss Jena, Germany) equipped with a 100x (planar) or 63x (apochromatic) oil-immersion objective and standard filters. Section of specific fluorescence were taken; the localization of tubulin and tubulin aggregates was visualized with FITC in green. Images were treated by multiplicative shading correction using software Axiovision (Zeiss Vision, Jena, Germany).
• CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14, 303-313.
• Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J. 77, 6124-6134.
• CD3 negative large granular lymphocytes recognize a heat-inducible immunogenic determinant associated with the 72 kDa heat shock protein (Hsp70) on human sarcoma cells. Blood 86, 1374-1382.
• Hsp70 heat shock protein
• Heat shock protein 70 stimulates proliferation and cytolytic activity of natural killer cells. Exp. Hematol. 27, 1627-1636.
• Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Natl. Acad. Sci. USA 77, 1561-1565.

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