UA103483C2 - Method for inducing cell death - Google Patents

Abstract

Provided herein is a method for inducing cell death by mducing heat shock response in a cell in combination with inhibiting adaptive heat shock response. Also provided are methods for preventing cancer, treating intracellular parasite infections, and inflammation - associated conditions.

Description

Cross-references to previous applications

ИО001) This application is based on prior US patent applications Mob1/054,785, filed May 2008, Mob1/102,913, filed Oct. 6, 2008, and Mob1/179,674, filed May 19, 2009, Table of Contents which is incorporated herein by reference in its entirety.

The field of technology

I0002| The invention relates to a method of inducing cell death by inducing a heat shock reaction in a cell in combination with inhibition of the adaptive heat shock reaction.

Prior art

I0003| Cancer is an acute problem of human health. It accounts for about a quarter of all deaths in the US and is the leading cause of death in men and women over 85. The lifetime probability of cancer is 4695 for men and 3895 for women. Most antiblastoma therapies have side effects, including nausea, vomiting, hair loss, fever, and risk of infection. Chemotherapy and radiation therapy quickly lead to inflammation of the mucous membranes of the oral cavity and gastrointestinal tract in patients, and these effects can be criteria for limiting doses of toxic drugs in some treatment processes.

I0004) Cancer cells show a high level of adaptive response to heat shock. This reaction is an adaptive mechanism used by all living organisms that have a cellular structure to survive in conditions of so-called proteotoxic stress, that is, a factor that leads to the accumulation of poorly structured proteins that are prone to aggregation, and the subsequent death of cells due to the complete denaturation of proteins , which is caused by such aggregation. Cells can activate the specified protective mechanism by inducing the synthesis of complementary proteins (chaperones), which are known as heat shock proteins (hereinafter referred to as HOR from the English words "peai 5posk rgoivip5"). 0005) The viability of cancer cells may be dependent on the heat shock response, as they have a higher level of protein structure disorders. However, the effectiveness of methods of heat therapy for cancer is limited by inducing an adaptive response to heat shock, which significantly reduces the sensitivity of cancer cells to therapeutic intervention and the possibility of using heat therapy and other methods of modulating heat shock. Accordingly, it is necessary to improve the methods of inducing cell death.

Brief summary of the essence of the invention

ИО00О6| A method of inducing cell death is proposed here, which consists of inhibiting the adaptive heat shock reaction in the cell and then inducing a heat shock reaction in it (further on in the abbreviation H5E from the English words "peai 5posK gezropze"). Such a reaction can be induced by introducing a heat shock inducer into the cell. Geldanamycin can be this inducer (further

OMAS17 or 17-OMAS or OM), proteasome inhibitor, arsenite or ethanol. A heat shock response can also be induced by increasing the internal temperature of the cell using a heating agent. The temperature can be increased to at least 39-60 C, in particular by using infrared radiation. This radiation can have a wavelength in the range of 5-15 µM. The temperature may also be increased by a heat source selected from the group consisting of an electroluminescent device, a laser diode, an emission laser with a vertical cavity surface, an LED, and a resistive incandescent lamp. The heat shock reaction inhibitor can be 9-aminoacridine (hereinafter 9AA) or carbazole (hereinafter С7 or SКВ2 or

SVI with a number).

ЙО007| The cell may be a cancer cell, and the cancer may be selected from the group consisting of metastatic breast cancer, bladder cancer, lung carcinoma, esophageal cancer, basal cell carcinoma, malignant melanoma, eye tumor, and head and neck cancer. The second method may involve inducing a heat shock response in the cell. For this, an antiblastoma agent is used.

Brief description of the drawings

IO0O8) Fig. 1 shows that acriquine (OS) and 9-aminoacridine (9ZAA) inhibit the synthesis of H5OR70 heat shock proteins in cells treated with proteasome inhibitors.

ИО009| Fig. 2 shows that OS and 9AA inhibit the transcription of the HOR7O gene in response to proteasome inhibitors.

IOO10) Fig. C shows that the synthesis of NZP70O induced by OMAS17 is sensitive to the action of OS and 9AA.

ИЙОО11| Fig. 4 shows that OS and 9AA inhibit the transcription of the H5P7OA1/B' gene induced by pMACI17 and the proteasome inhibitor MO132. 00121 Fig. 5 shows the toxic effect of OMASA17 in combination with OS on Her cells! and.

IOO13) fig. 6 shows the toxic effect of heat shock (43 "C, 30 minutes) in combination with OS. 00141) Fig. 7 shows that the sensitivity of cancer (ie, not normal) cells to heating increases under the influence of OS and C (compound SVI 0137).

IOO0O15) Fig. 8 shows that 5I-RNA-mediated reduction of EAST-nuclear complex, Z5PT1 and EAST-kinase SKII increases the sensitivity of cells to heating.

0016) Fig. 9 shows that acriquine and ZAA inhibit the synthesis of NZP7O in cells treated with proteasome inhibitors. Fig. ZA shows the results of immunoblotting of protein extract from cells

Nega, which were treated for 5 hours with the protease inhibitor MO132 (MO, 5 μM) in combination with the antimalarial drugs "emetine" (hereinafter EM, 1 μM), "chloroquine" (hereinafter CO), 20 μM), "quinine" (hereinafter

O, 20 μM) or "acrychin" (OS, 20 μM), with the use of anti-NZR7O antibody. Lane 1 contains an extract from untreated cells of Nei a. Pyrin expression was examined as a loading control (H.s.). Fig. 9B shows protein extracts from NeHa cells treated for 5 hours with the protease inhibitor MO132 (5 µM) alone or in combination with 20 µM OS or 9AA, after immunoblotting with anti-HeP70 antibody. Negative controls include untreated Nei cells! a (lane 1) cells treated with OS or 9AA in the absence of MO132 (lanes 6 and 7). Pyrin was studied as a load test (I..s.). Fig. 9C shows that emetine (but not OS or 9AA) inhibits overall protein synthesis. C55-labeled proteins from untreated NeHa cells (lane 1 on the left) and cells treated with OS (20 μM), 9AA (20 μM), or EM (1 μM) were examined by electrophoresis and autoradiography.

Fig. 90 shows that aminoacridines do not impair the proteasome inhibition by agent MO132, as shown by the results of proteasome activity assays using cell extracts

Nega treated for 4 hours with the indicated combination of the proteasome inhibitor MO132 (5 µM), OS (20 µM) and 9AA (20 µM). Proteasome activity is shown relative to its value taken for 100 95 in untreated cells. Fig. 9E shows that OS inhibits in cells No! and activation of transcription

H5ZR7OA1 proteasome inhibitors. 10 μg of total RNA isolated from untreated Nei a cells (lane 1) and cells treated for 5 hours with 0.1 μM bortezomib (hereinafter B) or 5 μM Mo132 (MO), taken alone or in combination with 20 μM OS were investigated by Northern blotting with NZP7OAT samples (upper panel) and SARON (lower panel) SARON was used to test the specific activity of OS as an inhibitor and RNA loading. Fig. 9E shows that aminoacridines do not interfere with the expression of the metallothionein gene under the influence of 2psCi2. Her a cells were untreated (lane 1 on the left) or treated for 5 h with 200 μM 7psIi2 (2n) alone or in combination with 20 μM OS or 9AA. 10 μg of the total mass of RNA was analyzed by Northern blotting with samples of metallothionein (hereafter MT), HER7OAT and SARON. 0017) Fig. 10 shows that the induction of the NOC heat shock reaction using MO132,

OMASI17 or heat shock as such is sensitive to aminoacridines. Fig. 10A shows the results of immunoblotting of a protein extract (10 μg) of NeHa cells treated for 5 hours with 5 μm

MO132 (MO) or 1 μM 17-OMAC (OM) alone or in combination with 20 μM OS or 9AA, using an anti-HOR7O antibody. Lane 1 contains an extract from untreated Neia cells. To check the load of proteins (I..c.), the expression of K18 was investigated. Fig. 108 shows that OS or 9AA inhibits the transcription of the НОР7OA1 gene induced by 17-ОМАС or MOо132. Northern hybridization

RNA from Her raw cells! a (lane 1) and the same cells treated for 5 hours with 1 µM 17-

OMA (OM) or 5 µM MoO132 (MO) alone or in combination with 20 µM OS or 9AA. SARON mRNA was used to test inhibitor specificity and RNA loading. Fig. 10C shows that OS and 9AA do not alter the accumulation of mRNA genes that were not induced by 17-OMAS, as shown

Northern hybridization of RNA from Nei a cells treated for 5 hours with 1 μM 17-UOMAO (CM) alone or in combination with 20 μM OS or 9AA. SARON, rbokeyA and SEROA4 are genes whose expression was not suppressed by the action of 17-OMAS. NOR7OA1 was included as a positive control for genes inducing 17-

OMA. Fig. 100O0 shows that OS inhibits NOE1-specific expression of the EEP generator induced by proteotoxic stress (hereafter the abbreviation H5НЕ1 from the English term "peai 5poskK

Tasiog" means "heat shock factor"). Nega cells, which are carriers of the H5E1- generator complex

ESRs were untreated (left bar) or treated with 0.1 μM bortezomib (82), 1 μM 17-OMAa (OM), or heat shock (H5) for 1 hour at 43 °C alone or in combination with 20 μM OS

The expression of ESR after 8 hours of exposure was examined with a fluorescence microscope.

IOO18) Fig. 11 shows that aminoacridines inhibit the activation of H5EI1 and its translocation to the nucleus.

Fig. 11A shows that the effect on cells of Not MOe132, 17-OMAS or heat shock induces the activity of binding NOT DNA. Cytoplasmic extracts from Neia cells were examined by electrophoretic mobility shift assay (abbreviation "EM5A"), and these cells were either untreated (left bar) or treated with 5 μM Mo132 (Mo) or 1 μM 17-OMAS (CM) for 4 h hours, or subjected to heat shock (43 "C) for 1 hour. The formation of a complex between NZE1 and a 32pP-labeled sample oligonucleotide having a H5E1 binding site was inhibited by a 50x excess of the same, but unlabeled, oligonucleotide having a site for MEKV binding (lane 4).

The specificity of the invented complex was further confirmed by its supershift in the presence of anti-H5E1 antibody (lane 7). Fig. 118 shows that OS does not reduce H5E1 DNA binding activity in the nucleus induced by proteotoxic stress. EM5BA was conducted as described in the explanations to

Fig. 11A, with labeled sample oligonucleotide and extracts from the nuclei of untreated cells of Her! a (lane 1 on the left), or treated for 4 hours with 5 µM Mo132 (Mo) or 1 µM 17-OMAS (SM) alone or in combination with 20 µM OS. Fig. 11C shows that bafilomycin increases the concentration of OS in the nuclei. Cells

Nega were treated with 5 μM OS alone or in combination with 0.5 μM bafilomycin for 1 h.

The intracellular localization of OS was investigated using an ultraviolet microscope and a blue filter. Fig. 110 shows that bafilomycin (hereinafter Vai) increases the activity of OS as an inhibitor of heat shock action. Her a cells were untreated (lane 1 on the left) or treated for hours with the indicated combinations of 5 µM MO132 (MO), 0.5 µM bafilomycin (Ba) and OS at various concentrations. Collected cell protein extracts were analyzed by Western blotting using an anti-NOZE1 antibody. Pyrin expression was examined as a loading test (.s.).

И0О0О19| Fig. 12 shows that OS enhances the cytotoxicity of inducers of proteotoxic stress.

Fig. 12A shows the toxicity of drug combinations as measured by cell viability testing. Cells

NeGha were treated for 4 h with the indicated combinations of 17-OMAS (1 μM) and OS (10 or μM). The viability of cells was determined after their 72-hour exposure by staining with methylene blue. The data shown are the average of three experiments. Fig. 128 shows the toxicity of the bortezomib/OC combination as determined by cell viability as indicated for FIG. 12A. Nae cells were treated for 4 hours with 0.1 μM bortezomib alone or in combination with 10 μM OS. The data shown are the average of two experiments. Fig. 12C shows immunoblotting data on apoptosis of NeHa cells treated with a combination of bortezomib (87) (0.1 μM), 17-OMAS (CM) (1 μM), and OS (10 μM). The markers were such apoptosis-specific proteolytic fragments as the complete protein РАКР and protein K-18 (РАКР/арори and К18/арори). Fig. 120 shows that H5P7O0-mediated 5i-RNA knockdown sensitizes cells to bortezomib and 17-OMAS, as determined by immunoblotting of proteins from HOR7O 5i-RNA of Neg cells treated with the indicated concentrations of 0.1 μM bortezomib (82), 20 μM OS, and 1 μM 17-OMAS (SM) and express 5i-RNA. Protein extracts were obtained from cells 24 hours after their transfection with HOR70 and treated overnight with bortezomib and 17-OMAS. Expression of H5OR7O and apoptosis-specific cleavage of K18 were identified with the appropriate antibodies. Control cells

Nega were untreated (lane 1) or treated overnight with drugs in the amounts indicated above (lanes 5-9). Fig. 12E shows that the caspase inhibitor, 2MAO-RMK, protects cells from apoptosis induced by a combination of proteotoxic stress and aminoacridines, as revealed by immunoblotting of HOP70 protein levels and apoptosis-specific cleavage of K18 in Her cells! a, which were treated overnight with the indicated combinations of 1 µM 17-OMAS (CM), 0.1 µM bortezomib (B2), 20 µM OS and 20 µM 2MA0-EMK (2MAB). Treatment of cells with anti-Ra5 antibody (Ra5, lane 9) and a combination of anti-Bach antibody and 2MAO-EMK (lane 10) provides, respectively, positive controls for apoptosis and the protective effect of 2MAO-EMK.

I0020) Fig. 13 shows the effect of 17-OMAS and acrichin on the growth of ip migo and ip mimo cancer cells.

Fig. 13A shows that OS suppresses the synthesis of H5P7O in response to proteotoxic stress of MCA2O5 and B-16 cells. RNA from control cells and cells treated for five hours with a solution of 1 μM 17-

RMAS (OM) in the presence or absence of 20 μM OS were analyzed by Northern hybridization in samples with

NBZR7OAT and SARON. Fig. 138 shows the results of tests on the survival of MCA20O5 and B-16 cells treated for four hours with a solution of 1 μM 17-UOMACE (CM) in combination with 20 μm OS.

Cells were harvested, diluted 1:50, and assayed for survival with methylene blue after 72 hours of incubation. Numerical values are average values for two experiments. Fig. 13C shows that intratumoral injection of OS blocks the synthesis of NZR7O induced by 17-OMAS. Mice line C57VI /6 as carriers of MCA205 tumors were subjected to a single intra-tumoral injection of phosphate-salt buffer solution (control), 25 μg of 17-OMAS (CM) or 1.25 μg of OSya25 μg of 17-OMAO. Five hours later, the mice were killed. RNA extracted from tumors was analyzed by Northern hybridization in samples with NB5P7OA1. Hybridization in samples with ZARON was used as a load check (i..s.).

Results obtained in two experiments are shown separately for each tumor with each treatment.

Figures 130 and 13E show the results of injections into mice of the C57VI /6 line with tumors of МСА20О5 (130) and B-16 (13E). Average tumor volumes for each group of mice are also shown.

I0021) Fig. 14 shows the antiblastoma effects of combining ineffective doses of acrichin and local hyperthermia in mice as models. 00221 Fig. 15 shows the effect of carbazole on HOR70 gene expression in response to proteotoxic stress, as determined by Northern hybridization of RNA isolated from control Her/a cells, cells treated for five hours with MO132 alone, a combination of MO132 with 10 μM 9AA, or 1 μm of carbazoles (SCVI) 159, 188, and 198 with a sample of the H5P70 gene. To check the load applied

SARON.

IOO23) Fig. 16 shows that SKV2A198 suppresses H5E1-specific expression of the SER generator. Cells

Nega infected with lentivirus expressing the generator gene were treated with 0.1 μM bortezomib (Bhi7) alone or in combination with 1 μM SKB2188 (inactive) and SKB2198 (active). CEP expression was determined 10 hours after the experiment using a microscope.

I0024| Fig. 17 shows the effect of heat shock (H5) and the combination of AA/SLE on NZE1-specific expression of SER. Nega cells infected with lentivirus expressing NOE1-specific CEP were exposed to H5 at 43 °C for 30 min, or H5 in combination with 20 μM 9AA, or 1 μM SKV2198 or

САВ159 (marking Ш/, i.e. "hypigeoid", indicates untreated cells Not a). SER was detected after 12 hours at 37 "С.

DETAILED DESCRIPTION OF THE INVENTION 0025) The inventors made the unexpected discovery that induction of the heat shock response in combination with inhibition of the adaptive heat shock response can cause cell death. Although not related to theory, inhibitors of the adaptive heat shock response may prevent the cell from exhibiting the ability to tolerate induction of the heat shock response by inhibiting the heat shock factor H5ET1, which is a major mediator of heat shock-induced transcription.

Heat shock proteins are chaperone proteins that prevent the fatal consequences of harmful stimuli in various ways by disaggregating, restructuring and renaturing damaged proteins.

The protective effect of heat shock proteins also lies in their ability to suppress some forms of cell death, including apoptosis. In the absence of heat shock proteins, cells are less able to respond to chemical, environmental and physiological stresses. In particular, cancer cells may be more sensitive to inhibition of the adaptive heat shock response because they normally express higher levels of heat shock proteins and may depend on those elevated levels as a mechanism to suppress the effects of cumulative mutations that would occur as a result of expression of deleterious proteins.

Accordingly, the combination of inhibiting the adaptive heat shock response and inducing the heat shock response provides a simple and flexible way to induce cell death. 1. Definition of Terms 0026) The terminology used here is intended only to describe certain embodiments of the invention and is not intended to be limiting. In particular, the terms used in the description and claims in the singular also denote the plural, unless the context clearly indicates otherwise.

I0027| To indicate numerical intervals here, each numerical parameter among others having the same measurement accuracy is presented explicitly. For example, in the interval 6-9, the numbers 7 and 8 should be considered along with the numbers 6 and 9, and in the interval 6.0-7.0 such numbers are 6.0:6.1; 6.2; 6.3; 6.4; 6.5; 6,6; 6,7; 6.8; 6.9 and 7.0. and. acrylic

I0028| As used herein alone or in combination, the term "acrylic" may refer to a radical

SnH2g-C(O)C(O)0-, where O is an aliphatic or aromatic group. r. acyl (0029) The term "acylamino" can mean K(-O)M-, where K is alkyl or aryl. with. introduction

IOOZO| As used herein, the term "administration" in relation to the dosage of drugs can mean either a single dose or a certain number of doses of a given drug. and. aliphatic

ИЙОО31| As used herein, the term "aliphatic" may refer to straight, branched, or cyclic hydrocarbon radicals that may or may not be substituted, saturated or unsaturated, but may not be aromatic. Further, the term "aliphatic" may include aliphatic groups containing oxygen, nitrogen, sulfur, or phosphorus atoms instead of one or more carbon atoms in the hydrocarbon chain. e. Alkoxyl

II0032| As used herein alone or in combination, the term "alkyl" may refer to alkyl, alkenyl, or alkynyl attached by a simple terminal ether bond. Examples of methoxyl groups can be methoxyl, ethoxyl, n-propoxyl, isopropoxyl, n-butoxyl, 2-butoxyl, tert-butoxyl, n-pentoxyl, 2-pentoxyl, 3-pentoxyl, isopentoxyl, neopentoxyl, n-hexoxyl, 2-hexoxyl , 3-hexoxyl, 3-methylpentoxyl, pfluoromethoxyl, difluoromethoxyl, trifluoromethoxyl, chloromethoxyl, dichloromethoxyl and trichloromethoxyl. "Alkoxyl" also means -OK, where K is alkyl.

Y. Alkoxyalkyl

100O33) The term "Alkoxyalkyl" means an alkyl group in which the hydrogen atom is replaced by an alkyl group. 9. alkenyl

I0034| As used herein alone or in combination, the term "alkenyl" may refer to a branched or unbranched unsaturated aliphatic group containing at least one carbon-carbon double bond at a specified location along the chain. Typical examples of an alkenyl group include ethenyl, E and 7 pentenyl, decenyl, and the like.

J. alkyl 0035) As used herein alone or in combination, the term "alkyl" may refer to a straight or branched chain hydrocarbon group containing a certain number of carbon atoms, for example: methyl, ethyl, straight or branched propyl or butyl. and. alkylthio

I0OOZ6) The term "alkylthio" can mean -9K, where K is alkyl.

). alkynyl

I0037| As used herein alone or in combination, the term "alkynyl" may refer to a branched or unbranched unsaturated aliphatic group containing at least one carbon-carbon triple bond at a specified location along the chain. Typical examples of an alkenyl group include ethynyl, pentynyl, propargyl, and the like.

K. amino

ИЙОО38| As used herein alone or in combination, the term "amino" may refer to a group containing a nitrogen atom in the chain. Typical (but not limiting) examples of amino groups include alkylamino-, dialkylamino-, arylamino-, diarylamino-, alkylarylamino-, alkylcarbonylamino-, carbamoyl, ureido-, and the like. The term "amino" as such means -MNaI2, and the term "alkylamino-" means -MH2, where one K is alkyl and the other K is an alkyl or hydrogen atom.

I. aromatic

ИО0О39| As used herein alone or in combination, the term "aromatic" may refer to an unsaturated hydrocarbon group having 4p-2 unlocalized p(r) electrons and may be substituted or unsubstituted. Further, the term "aromatic" may refer to aromatic groups containing at least one nitrogen atom instead of one or more carbon atoms in the hydrocarbon structure. Typical examples of an aromatic group are phenyl, naphthyl, thienyl, furanyl, pyridinyl, (is)oxazoyl, and the like. t. alkylsulfinyl 00401 The term "alkylsulfinyl" means K-502-, where E is alkyl. n. alkylsulfonyl 00411 The term "alkylsulfonyl" means K-503-, where E is alkyl. at. morpholine group o (0042) The term "morpholine group" means with . r. tetrahydrofuryl group and 0043) The term "tetrahydrofuryl group" means a. piperidinyl group' (0044) The term "piperidinyl group" means C or aryl 0045) The term "aryl" as used herein alone or in combination may refer to a substituted or unsubstituted aromatic group optionally attached to another aromatic or non-aromatic cyclic group . Typical examples of the aryl group are phenyl, methylphenyl, naphthyl, phenylene, phenylidene, xylyl, styryl, phenethyl, benztriyl, and the like. 5. aryloxyl

I0046| As used herein alone or in combination, the term "aryloxy" may refer to an aryl group attached by a simple terminal ether bond.

I. branched

I0047| As used herein alone or in combination, the term "branched" can refer to a group containing from one to 24 bonded atoms and having in the main chain at least one or more subordinate chains. A branched group can contain from one to 12 basic atoms.

Examples of branched groups: isobutyl, tert-butyl, isopropyl, -СН2гСН2СнН(СНЗСН2СН2-, -СН2 сн(исн2генЗСн2еСНО-, -СН2СН2гС(СНЗ)З. i. рак

I0048| As used herein, the term "cancer" may refer to any state of resistance to apoptotic stimuli. m. cancer therapy

I0049| As used herein, the term "cancer therapy" may refer to any method of cancer treatment known in the art, including chemotherapy and radiation therapy. m. carbonyl

I0O50| As used herein, the term "carbonyl" or "carboxyl" may mean any group containing a carbon-oxygen double bond. Typical (but not limiting) examples of carbonyl-containing groups are aldehydes (i.e. formals), ketones (i.e. acyls), carboxylic acids

(ie carboxyls), amides (ie compounds with amido groups), imides (ie compounds with imido groups), esters, anhydrides and similar compounds. h. combining

ИЙ0ОО51| As used herein, the term "combination with" or "concomitant use" to describe the use of aminoacridine and adjunctive therapy may mean that aminoacridine is administered before, during, or after the adjunctive therapy, or a combination thereof. in. cyanogen

As used herein alone or in combination, the term "cyan", "cyanate" or "cyanide" may refer to a carbon-nitrogen double bond. Typical (but not limiting) examples of cyano groups are isocyanates, isothiocyanates, and the like. The term "cyan" means -CM. 7. cyclical

І0052| As used herein alone or in combination, the term "cyclic" or "cyclo" may refer to a radical having one or more closed saturated or unsaturated rings formed of 3-12 (or 3-7) primary atoms. ah effective amount

I0053)| The term "effective amount" as used herein with respect to a compound, product, or composition may mean such an effective amount of the compound, product, or composition as to achieve the desired result. The exact amount required will vary depending on the particular compound, product or composition used, route of administration, etc. Thus, it is not always possible to precisely determine the "effective amount" in advance. However, an acceptable effective amount can be determined by one of the methods known to those skilled in the art through routine experimentation alone. yr.haloalkyl (0054) The term "haloalkyl" means an alkyl substituted with one or more, for example from 1 to 3, halogen atoms, in particular fluorine, chlorine, bromine, iodine or combinations thereof. Similarly, "halocycloalkyl" is a cycloalkyl group containing one or more halogen substituents. 0055) The term "aryl" alone or in combination means a monocyclic or polycyclic (preferably monocyclic or bicyclic) aromatic group, for example, phenyl or naphthyl. Unless otherwise specified, "aryl" may be unsubstituted or substituted, for example, by one or more, particularly from one to three halogen atoms, alkyls, hydroxyalkyls, alkoxyls, alkoxyalkyls, haloalkyls, nitro-, amino-, alkylamino-, acylamino- , alkylthio, alkylsulfinyl, and alkylsulfonyl groups. Examples of aryl groups are phenyl, naphthyl, tetrahydronaphthyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2-methylphenyl, 4-methoxyphenyl, 3-trifluoromethylphenyl, 4-nitrophenyl and the like. ss halogen 0056) As used herein alone or in combination, the term "halogen", "halide" or "halo" means fluorine "B", chlorine "CI", bromine "Bi", iodine "I", and astat "A". Typical examples of halogen-containing groups are chloracetamide, bromoacetamide, iodoacetamide and the like. aa. hetero

I0057| As used herein in combinations, the term "hetero" means one or more atoms of any element other than carbon or hydrogen. Typical examples are heterogroups containing nitrogen, oxygen, sulfur and phosphorus as heteroatoms. eh heteroaryl (0058) The term "heteroaryl" means a monocyclic or bicyclic ring system having one or two aromatic rings and containing at least one nitrogen, oxygen or sulfur atom in the aromatic ring and which may be unsubstituted or substituted, for example with one or more, in particular, one or three substituents such as halogen, alkyl, hydroxyl, hydroxyalkyl, alkyl, alkoxyalkyl, haloalkyl, nitro-, amino-, alkylamino-, acylamino-, alkylthio-, alkylsulfinyl-, and alkylsulfonyl- groups. Typical (but not limiting) examples of heteroaryl groups are thienyl, furyl, pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazonyl, and thiadiazolyl.

I0O59| The term "alkylene" means an alkyl group having a substituent. For example, the term "Si-

Alkylenaryl" refers to an alkyl group containing from one to three carbon atoms and substituted by an aryl group. its. heterocycle

As used herein, the term "heterocycle" may refer to a cyclic group containing a heteroatom.

Typical (but not limiting) examples of heterocycles include pyridine, piperidine, pyrimidine, pyridazine, piperazine, pyrrole, pyrrolidine, pyrrolidinone, morpholine, thiomorpholine, indole, isoindole, imidazole, triazole, tetrazole, furan, benzofuran, dibenzofuran, thiophene, benzotriazole, benzoxazole, benzthiophene, quinoline, isoquinoline, azapine, naphthopyran, furanobenzopyranone and the like. yes heterocycloalkyl

I0061| The term "heterocycloalkyl" means a monocyclic, bicyclic and tricyclic cycloalkyl group containing in the ring one or more heteroatoms selected from the group consisting of oxygen,

nitrogen and sulfur. A heterocycloalkyl group may also have an oxo group (O) attached to the ring.

Typical (but not limiting) examples of heterocycloalkyls are 1,3-dioxolane, 2-pyrazoline, pyrazolidine, pyrrolidine, piperazine, pyrroline, 2H-pyran, 4H-pyran, morpholine, thiopholine, piperidine, 1,4-dithiane, and 1,4 -dioxane.

PAS. hydroxyl 00621 The term "hydroxyl" means -OH. ii. hydroxyalkyl

100O63) The term "hydroxyalkyl" means an alkyl group in which hydrogen is replaced by hydroxyl. ). lower

I0064| As used herein, the term "lower" may refer to a group having from 1 to 6 basic atoms.

KK. nitro

I0065| The term "nitro" means -MO2.

I. at will 0066) The term "at will" or "desirable" used here may mean that the description of cases that follows it includes various possible options, which are distinguished by the presence or absence of some feature. For example, the phrase "optionally substituted alkyl" can mean that an alkyl group can be either substituted or unsubstituted, and that such a definition includes both options. pp. peptide

II0067| A "peptide" or "polypeptide" is a chain sequence of amino acids and can be natural or synthetic or a combination of natural and synthetic. pp. phosphate-containing group

IOO68| As used herein, the term "phosphate-containing group" may mean a group containing at least one phosphorus atom in the oxidized state. Typical (but not limiting) examples of compounds with such groups are phosphonic acid, phosphinic acid, phosphate esters, phosphinidene, phosphine, phosphinyl, phosphinylidene, phosphoranyl, phosphoranilidene, and the like. this saturated

I0069| As used herein, the term "saturated" may refer to a group in which all available valences of the main atoms are locked to other atoms. Typical examples of saturated groups are butyl, cyclohexyl, piperidine and the like. yr replaced

I0070| As used herein, the term "substituted" may mean a group having one or more hydrogen atoms removed from a carbon atom or heteroatom and replaced by other groups. Substituted groups can have from one to five or from one to three substituents. An atom with two substituents is called "di" or "di" while an atom with more substituents is called "poly" Typical examples of such substituents are aliphatic groups, aromatic groups, alkyl, alkenyl, alkynyl, aryl, alkoxy, halogen, aryloxy, carbonyl , acrylic, cyano, amino-, nitro-, phosphate-containing groups, sulfur-containing groups, hydroxyl, alkylcarbonyloxyl, arylcarbonyloxyl, alkoxylcarbonyloxyl, aryloxycarbonyloxyl, alkylcarbonyl, arylcarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, acylamino, amidino, arylalkyl, imino , тіокарбоксилат, алкілсульфініл, трифлуорметил, азидо, гетероцикліл, алкіларил, гетероарил, семикарбазид, тіосемикарбазид, малеїмід, оксіміно, імідат, циклоалкіл, циклоалкілкарбоніл, діалкіламіно, арилциклоалкіл, арилкарбоніл, арилалкілкарбоніл, арилциклоалкілкарбоніл, арилфосфініл, арилалкілфосфініл, арилциклоалкілфосфініл, арилфосфоніл, арилалкілфосфоніл, арилциклоалкілфосфоніл , arylsulfonyl, arylalkyl sulfonyl, arylcycloalkylsulfonyl or combinations thereof and their substituents. yes sulfur-containing group

I0071) As used herein, the term "sulfur-containing group" may mean a group containing a sulfur atom.

Typical (but not limiting) examples are sulfhydryl, sulfene, sulfin, sulfinyl, 5-thio-, sulfonyl, thio-, thioxo-, and the like groups. gg treatment and treatment 00721) As used herein, the terms "treatment" and "treatment" when referring to the protection of mammals from harm may mean the prevention, suppression, repression or elimination of harmful factors.

Prevention means prophylactic treatment of mammals before the onset of harmful factors. Suppression means the treatment of mammals after the onset of harmful factors, but before the appearance of clinical manifestations of the disease. Repression means the treatment of mammals after the occurrence of clinical manifestations of the disease in order to weaken or stabilize the effects of harmful factors. Eliminating the effects of harmful factors means treating mammals after the onset of clinical manifestations of the disease in order to relieve them of suffering. 55. trifluoromethoxyl

I0073| The term "trifluoromethoxyl" means -OSEZ.

NO. trifluoromethyl

I0074| The term "trifluoromethyl" means -SEZ.

ci cancer cell

I0075| As used herein, the term "cancer cell" may refer to any cell that is resistant to apoptotic stimuli. mm unbranched

I0076| As used herein, the term "unbranched" may refer to a group having from 1 to 24 primary atoms where the main chain or backbone extends in a straight line. An unbranched group can contain from 1 to 12 basic atoms. Jr. unsaturated

I0077| As used herein, the term "unsaturated" may refer to a group where at least one available valency binding two adjacent main atoms is not locked to another atom. Typical examples of unsaturated groups are -"СНЕСН.СН-СН", phenyl, pyrrole and the like. xx not replaced

I0078| As used herein, the term "unsubstituted" can mean a group that does not have any attached or substituted group. 2. The method of inducing cell death

І0079| A method of inducing cell death is proposed here. Such death can be induced by inhibiting the adaptive heat shock response in combination with inducing this response in the cell. The heat shock reaction can be induced by introducing a suitable inducer agent into the cell or by increasing the internal temperature of the cell with a heating agent. and. Inhibition of the adaptive response to heat shock

I0OO801 An inhibitor of the adaptive response to heat shock can be a non-genotoxic agent capable of being incorporated into DNA, which suppresses H5ZE-1-mediated transcription. This inhibitor of the adaptive response to heat shock can be aminoacridine, carbazole, or its derivatives. (1) Aminoacridine

I0081)| Aminoacridine can have the following formula:

Burrows

CE "V. ovi

IN; MO V. (Compound 1) de

C: is H or halogen;

Ve is H or, optionally, alkyl;

Az is H or, optionally, alkyl; and

KA is H or, optionally, an aliphatic group, aryl, or heterocycle.

I0082| The aminoacridine can be 9-aminoacridine or Merasgipe, which is also known as acriquine.

The aminoacridine may be as described in UMO/06/12419, the content of which is incorporated herein by reference. (2) Carbazole derivatives 0083) An inhibitor of the adaptive heat shock response can be carbazole or its derivatives.

Carbazole or its derivatives can be as described in Ch.5. Rgomysiope! Arrisaiop 61/102,913 dated Oct. 2008, the contents of which are incorporated herein by reference. (0084) Carbazole or its derivatives can have the formula (1): o in vo

D TE

V" M V

E 1 I. E and

Vl (snvK carries" ve V. vi is)

where

Be is selected from the group consisting of hydrogen, C:-alkyl, C:-vehalogenoalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OB8, M(Be)2 and ZKU, or Ka and B" or ME? and KK" together with carbon atoms to which they are attached and create a five- or six-membered aliphatic carbocyclic or heterocyclic ring;

IN? selected from the group consisting of hydrogen, C:-alkyl, C1-haloalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OKe, M(Be)2, and ZK, or K? and K5 or MK: and Ke together with the carbon atoms to which they are attached and create a five- or six-membered aliphatic carbocyclic or heterocyclic ring;

AB: selected from the group consisting of hydrogen, C1-alkyl, C1-hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and C(-O)B2, or VE: and RU, which together form five-, six- , or a seven-membered aliphatic ring optionally containing an oxygen atom;

VU is selected from the group consisting of hydrogen, C1-alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and C(5O)B", or BZ and B" together with the carbon atoms to which they are attached and create five- or a six-membered aliphatic ring;

Bhe is independently selected from the group consisting of hydrogen, C1-alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, or two NC: groups which, together with the nitrogen atom to which they are attached, form a five- or six-membered aliphatic ring ;

B', Be, BAZ, B, P» and KU are independently selected from the group containing hydrogen, C:-alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halogen, OK. Сb(-0)Не, Сб(-0)0Не, "С(-О)Ke, С(-О)М(Вг)",

Сб(-О0)MAeBOgve, M(Не)», MAеС(-О)Не, MAеС(-О)М(Не)», СМ, MO», СЕз, OSЕз, 5Не, 5ОВе, 5022,

ЗО2М(Ве)", and О5О»СЕ with;

A" is selected from the group containing hydrogen, C1-alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl; pe0,1,2,3,4, or 5, or a pharmaceutically acceptable salt or hydrate thereof. 0085) Carbazole or its derivatives can have the general structural formula (Ia): o EE o

Ts and v i Still Wed

In M Vo in ! vi (Sheaf 1 and not vve (a) de

Ve is C1-alkyl, C1-haloalkyl, C3-cycloalkyl, M(K-)2, or OKU, or Kg and 2! together with the carbon atoms to which they are attached and create a five- or six-membered aliphatic carbocyclic ring;

Is it C3-alkyl, C1-haloalkyl, C3-cycloalkyl, M(K-)2, or OK, or EC? ЭЯ together with the carbon atoms to which they are attached and create a five- or six-membered aliphatic carbocyclic ring or a five- or six-membered aliphatic carbocyclic ring containing a nitrogen atom;

C: is C1-alkyl, C3-cycloalkyl, or C1-hydroxyalkyl;

VU is hydrogen, C:-4alkyl, or C3"cycloalkyl, or KU and B" together with the atoms to which they are attached and create a five- or six-membered aliphatic ring containing a nitrogen atom, or B: and RU, which together create a six- or seven-membered aliphatic ring containing, if desired, an oxygen atom;

Be is independently hydrogen or C. -alkyl;

B" is hydrogen or C alkyl;

E is hydrogen, hydroxyl, or C.i.-alkoxyl;

BZ and 87 are independently hydrogen or C-alkyl;

IN? is hydrogen, hydroxyl, C: zaloxyl, or halogen;

Ve is hydrogen, C1-alkyl, C1-alkoxyl, or halogen;

B" is hydrogen or C alkyl; it is 0,1,2,3,4, or 5,

or a pharmaceutically acceptable salt or hydrate thereof. (0086) Carbazole or its derivatives may have the general structural formula (IB):

Ov o

N c

In M vo ha | In (sno)a net ve "vi chu de

Ve is methyl, ethyl, n-propyl, cyclopropyl, MH(CH»3), or OSN», or Wag and E! together with the carbon atoms to which they are attached and create a five-membered aliphatic carbocyclic ring;

Ve is methyl, ethyl, n-propyl, cyclopropyl, MH(CH3), or OCH", or P? and Re together with the carbon atoms to which they are attached and create a five-membered aliphatic purely carbocyclic ring or a five-membered aliphatic carbocyclic ring containing a nitrogen atom;

B: is methyl, ethyl, n-propyl, isopropyl, cyclobutyl, or 2-hydroxyethyl;

VU is hydrogen, methyl, ethyl, or cyclobutyl, or KU and B" together with the carbon atoms to which they are attached to form a five-membered aliphatic carbocyclic ring containing a nitrogen atom, or

В: и КУ, which together create a morpholine group, a tetrahydrofuryl group, or a piperidinyl group k or a group;

B" is hydrogen;

A: is hydrogen, hydroxyl, or methoxyl;

VZ is hydrogen;

B: is hydrogen, hydroxyl, methoxyl, or fluorine;

Ve is hydrogen, methyl, methoxy, or fluorine;

B" is hydrogen; n is 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof.

I0087| Carbazole or its derivatives can have the structural formula (I): and others

OM ik i-. MO" to (A M (A in and Y in di (SN vya

And fe their shchi "on , Fo de

B is selected from the group consisting of hydrogen, C1-alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and

C(-O)B", or B/ and 5, which together create a five-, six- or seven-membered aliphatic ring containing, if desired, an oxygen atom;

Ve is selected from the group containing hydrogen, Ci-e alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl and C(5O)B", or z and KE", which, together with the atoms to which they are attached, create five- , a six- or seven-membered aliphatic ring;

B" is independently selected from the group containing hydrogen, C-C alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, or two P" which, together with the nitrogen atom to which they are attached, form a five- or six-membered aliphatic ring ;

VU, VO, VA", Ve, A, and B"? independently selected from the group containing hydrogen, C:-alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halogen, OK", C(-0)8", C(-0)OVA", C(O)v C( -OM(B",

С(-О)MA"BO»A", M(VG)2, MA"С(-О)А8", MA"С(-О)М(А")», SM, MO», SEz, OSEz , 58", BOV", 502",

ZOV)" and O5OSE:.;

VAZ is selected from the group consisting of hydrogen, C1-alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl; reb,1,2,3,4, or 5, provided that when p is 2, one of B. and 29 is other than ethyl, or a pharmaceutically acceptable salt or hydrate thereof. (0088) Carbazole or its derivatives can have the general structural formula (Pa): da dz

Sam, : MO to (I m g dz ; | , "Shen She shchi shY

M

But also shu "Sho de

V' is S. valkil;

B» is hydrogen or C3-alkyl, or Kz and E8 which, together with the atoms to which they are attached, create a five- or six-membered aliphatic ring containing one nitrogen atom;

B" is hydrogen or C alkyl;

B" is hydrogen, hydroxyl, or C:-alkoxyl;

A" and B": independently is hydrogen or C -alkyl;

B"Z is hydrogen, hydroxyl, C: zaloxyl, or halogen;

B": is hydrogen, C-zalkyl, or C: zalkoxyl;

VU is hydrogen or C alkyl; ed,1,2, 3,4, or 5, provided that when p is 2, one of B! and EC: is other than ethyl, or a pharmaceutically acceptable salt or hydrate thereof. 0089) Carbazole or its derivatives may have the structural formula (NAME):

SM. In V and MS

Her widow - NV de

B' is methyl or ethyl;

Ve is hydrogen or methyl, or Kz and Ku, which, together with the atom to which they are attached, create a five-membered aliphatic ring containing one nitrogen atom;

B is hydrogen; p is 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof.

I0090| Carbazole or its derivatives may have a structural formula selected from the group consisting of: sk , -d-O m ; with SNaz. i ach M

SkM | MO s MO» sh s dk MN

SNU and the same sgry po, about ah m i sov / a and even more about TSO go; 7 and Y Her and TSI EH what? o E Eh I / : / more More with | catfish tki MO rochoya u

In sh: o ho: i and Uh!

AD E Her. ; and these ra: . y - t 4 : o / o /

And" MN

SN I with Sn o go: o (o i ! u. і da Y h I o do che - I mae do Y / - no / She a:

MA UNN and what

Shhhh oh oh oh

I Y S her ud - y soe u y | For Fr

M EK

ЧЧЧЧ

SN o Z o o;

U. in N

Useguh ostu" / y s ME F m F o NIV: what and (A shchi i tm ( " y Z M. y "/

Z Z s o ii ii ii ii what | teh i shvi F ri ak wa sche o; about in and yi yi yi

So rea g» g en

M We

Oh he

WITH

. o Z o o Her Y

I and rep i (I 7 " "

M K- and more

Z o o a : M y o o, o o o o o them | and

On pozodaovovonoy

X i 4 thick oil | I and AL a shk i z mod sho toneto onov shi sho ssh pt,

All Udoo is about this be | Yo ooo oh oh

Ge) y z o i Y she va Ж plo re Ж 7 B (l cho m V - i s n h | Ya she, tyah i ich ne yo z ich S ! z | z. x a (FI o and other chn : o aa and yours is not S shcha No x NS s y what tt - g

AJ And that, oh oh Z

It is also, ch i a u y y: x g st B y z "A personal eotnya is not sown; Noi and

Z y ge yi i r a t

Хі І І і ra a Е Z i p a ; wai - par chn pet. another 3rd floor; pig: wow, we still:

B also Z

How else,

From g; With o ut i and . just 3 g! b j Shk shcha v i z u u o o o o GI o I c) i ; eat

Nam h. ch. shi sho sho M

AND

; ; and

This is Ya She sna sn pu oh oh I pceo

C | t se z i 7 CH N

ОН 4 pb I i i Ii i

IY m i h o / sho i

M M i s ( still s Y sn and Sma Ne ,

On sne and yknh, o u z x x | no

KAH ra A S y - y ( / it is Y y sh ay NO, dn y sne | nya , sne ,

si so ; h o o boro aka oman 4 om tn ome

Also and and

M shcha g and z | Z I .e gav) a ; about what m

M M I what U, We

To me and to me. G » cm MO

SO esutu" som MO Ot

WITH

; i sleep

M and | nothing

M I

OM is MO

Somy schu m s Y /

M s m o, | yz g sna o acha o Ia 8 -schy bi bi yerror'usyu Su still .

Ko) oh oh with her and more; | M! M / / MN " ( g Z , sne gav; o ich Y g ro ch her o, and Ashchsche S - M yo Ibf" - in p lk |. re / Io ! NU M y Sya

M

MH;

Sleep Zh and m. g

GU ho it, ; ko D) C) /y z U NN o o tuh sha: ЧИ щ m ze I

With ss / ) in

MN and

Mo

: ME sn: shi sho ; z z sini G Mo ii: z Z IF ii ! . ; "oh -- ! all I am! and | y Z Be A ME shin ; And / x that in

M t ZI y Z «fo --s , ON y Sh , o I I o I - i iii shu Her i vah k o a So. at

І І ИЗ | and

Sho (Я со 0007 | ше: и ији Фи С : m sy re С M ч (А чо С "

M M

, in, S , o o : i

And still eat

NM still.

She and-y have him. , o o sh-o r ns

And, in ; M a c) o ee lo-

I0O091| Carbazole derivatives can have the following structural formula:

Ж -7 ke re

GA, F

IS . 0092) Carbazole may also be selected from the group consisting of 3:

SVI 0137

He, no

OZ o iIzhko 7-no mch Sho i sin i"

SN.

SVI 0197 -4 de av,

M and SIN

Wed, Nov 0198

J

- be g SIN and not all 100

YES and

And still sleep, SV,

SVI 0159

SYA, y

M

/y sin what

Sun, Sun 0212

SN, Snika! p-o a "M

Fe 7 ON

SVI 0174

SN. but before Bech.

I still have SIN

I. t and SVI 0175 sn. as

And or about I "F,

KI I sleep. (3) Inhibitor of transcription or translation

I00931| An inhibitor of the adaptive heat shock response can also be an inhibitor of transcription or translation. For example, it can be actinomycin O or cycloheximide. r. Heat shock-inducing agent or factor (1) Heat-shock-inducing agent 0094) The heat-shock response in a cell can be induced by a heat-shock-inducing agent.

This agent can be such an inhibitor of protein synthesis as puromycin or azetidine. This agent can also be a NOROO inhibitor such as geldanamycin (OMAS17), radicicol, or 17-AAO. This agent can also be a protease inhibitor such as Mo132, bortezomib or lactacystin. This agent can also be such an inhibitor of serine protease as OSIS, TRSK, or TI SC. This agent may be an inflammatory mediator such as cyclopentenone, prostaglandin, arachidonate, or phospholipase A." This agent can also be a triterpenoid such as celastrol. This agent may be a co-inducer such as a non-steroidal anti-inflammatory agent such as sodium salicylate or indomethacin, or a hydroxylamine derivative such as bimoclomol or arimoclomol. This agent can also be a flavonoid such as quercetin or a methylphenylidene lactam compound of the KMK437 type. This agent can also be arsenite or ethanol. (2) Heating means

Y0095| The heat shock reaction in the cell can also be induced by a heating agent.

Heating means can be photoelectric, mechanical or chemical. Heating means may be capable of non-invasively heating the cell, in particular through the skin. The heating means may also contain a wire, lumen, receiver, probe or catheter as described in SO.5. Paradise Ryb. Me20070288075.

Heating devices may contain optical fibers, filaments, or an implanted device. The heating means may be as described in US Patent No. 5,814,008, the contents of which are incorporated herein by reference.

ИО096| The heating means may also contain a sensor to measure the temperature of the cell or tissue in the treatment area and, accordingly, generate a signal indicating the temperature. Temperature can be measured with a thermocouple, resistance thermometer, or thermistor. The sensor can be in the form of a needle or a probe. The temperature of the heating medium can be adjusted according to the specified signal.

The temperature can be adjusted to maintain it at a predetermined level or to prevent the cell or tissue being treated from overheating due to exceeding a predetermined level by varying the amount of energy used to power the heating means. (a) Photoelectric heaters

I0097| The heater can be such a light source as a set of solid light-emitting devices. For example, the light source can be an LED, an electroluminescent device, a laser, a laser diode, a laser with a vertical cavity, or an incandescent lamp. Light can be visible, near-infrared, or infrared. For example, the wavelength can be from 750 nm to 1 mm. The wavelength can also be 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970 , 980, 990, or 1000 nm. The wavelength can also be 1;1,151,2:1,351,4:51,5;1,6;51,7;1,8;51,952;2,1;2,2:2,3;52,452,5;52 ,6;52,752,8;52,9; 3; 3.1; 3.2; 3.3; 3,4; 3.55 3.6; 3.7; 3,8:3,954:541:42:4,3:44:4,5:54,6:4,7:54,8:54,95555,1;5 52:53; 5.4; 5.5; 5.6; 5.7; 5,8:55,9:56;6,1;6,2;:6,3:6,4:56,5;56,6;6,756,8:6,9:57571;572:7,3 ;7,A:57,557,6;7,7;57,8;7,9:58:8,1; 8.2; 8.3; 8,4:8,5:8,6:;8,7:8,8:58,9:59:59,1:592;:9,3:59,4;:9,5:59,6 :59,759,8:59,9;10;10,1;102;10,3;10,4;10,5; 10.6; 10.7;10.8;10.9511;11.1;11.2:511.3511.4:511.5511.6;511.7511.8;11.9:512;:12.1;12 ,2; 12.3; 12.4; 12.5; 12.6; 12.7; 12.8; 12.9; 13; 13.1; 15.2: 13.3; 13.4; 13.5; 13.6; 13.7; 13.8; 13.9; 14; 14.1; 14.2; 14.3; 14.4; 14.5; 14.6; 14.7; 14.8; 14.9; or 15 µM. The wavelength can also be 1,2,3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 , 99, or 100 µM. The wavelength can also be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 . . ,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980 , 990, or 1000 Rp.

I0098| The light source can be an intense laser, in particular, using the two-photon method. The laser can emit a well collimated beam. The light source can also be weak and incoherent. Light can be emitted starting from a few photons per square meter (ie, have a low power density compared to a high-intensity laser).

For example, the energy can be 30-25000 J and the power density can be less than 500 mW/cm.

I0099| The heating means may emit microwave or radio frequency energy as described in US Patents Mob,904,323 and Me5,549,638, the contents of which are incorporated herein by reference.

Heating means can have an antenna, in particular in the form of a grid. Heating devices can also have an ultrasonic transmitter. (p) Mechanical heaters

IOTO0I| Heating means can have a resistance element. This resistance element can be an incandescent lamp. The heating means may also have a heating liquid or gas placed in a cylinder, as described in Ch.5. Paradise Rir. Mo. 2007/0288075, the contents of which are incorporated herein by reference. (c) Chemical heaters

ИО101|) The heating agent may contain a chemical agent capable of increasing the internal temperature of the cell. For example, the chemical reagent may contain iron oxyhydroxide particles with dextran or an iron complex. The chemical reagent can also be gallium, indium, technetium, strontium, iodine or other material compatible with a living cell. A chemical agent can increase the internal temperature of a cell by accelerating metabolism or by its own oxidation in the cell to increase blood oxygenation levels, as described in US Pat. No. 4,569,836, the content of which is incorporated herein by reference. (4) Temperature

IO102| The internal temperature of the cell can be increased to at least 39, 40, 41, 42, 43, 44, 45, 46, 47, A8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 "C. The internal temperature of the cell can also be increased in the range of 1-38 "C, 39-45 "C, 45-60 "C, 60-85 C, or 85-100 "C. The temperature should be sufficient to damage or kill the aminoacridine-treated cell, but below the level at which damage or death of the healthy cell begins, that has not been treated The temperature must also be sufficient to induce heat shock The internal temperature of the cell can be raised for at least 1, 2,3,4,5,6,7,8,9,10, 11, 12, 13 , 14, 15,16, 17, 18, 19,20,21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, Ab, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 x Vylyn, or for the time 1,2,3,4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, A8 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 hours. with. Introduction

IO103) The compounds, chemical reagents and agents described here can be introduced into the cell by absorption and diffusion into the patient's cardiovascular system. Administration can be oral, parenteral, transdermal or using suppositories. Administration into the cell can be done with a needle attached to a syringe, or a catheter attached to an intravenous infusion system, or locally. 3. Treatment of the patient 0104 The method provided here can be used to treat a disease or eliminate the effect of a harmful factor in a patient in need. An inhibitor of the adaptive heat shock response can be administered to the patient and the heat shock response can be induced in the patient locally or as a whole. For example, an agent capable of inducing a heat shock response can be introduced into a cell such as a cancerous or infected cell. A heat shock response inducing agent can be administered to the patient as a whole. The internal temperature of the cell can be increased by the local effect of a heating agent on the cell. The patient's body temperature as a whole may also be elevated, but only up to physiologically acceptable hyperthermia. and. Cancer

ИЙО105| Cancer or precancerous conditions can be treated in the manner disclosed herein. Cancer can be a tumor located under the skin or on the skin. The tumor can be small or large.

Possible brain cancer, including glioma, glioblastoma, astrocytoma, anaplastic astrocytoma, astrocytoma-oligodendroglioma, pituitary adenoma. Pulmonary or pleural mesothelial cancer is also possible, such as lung cancer, lung carcinoma, non-small cell lung carcinoma, tracheobronchial cancer, early stage lung cancer, endobronchial metastatic cancer, peripheral lung cancer, or malignant pleural mesothelioma.

IO106| Gastroenterological cancer is also possible, as a pre-malignant or malignant tumor located inside the esophagus, bile duct or colorectum. Head and neck cancer, esophageal cancer, early stage esophageal cancer, Barrett's esophagus, basal cholangiocarcinoma, hardening of the liver or pancreas, liver metastases from colorectal cancer, or pancreatic cancer are also possible.

ЙО107| Cancer can also be a urological disease such as recurrent papilloma, bladder cancer, superficial bladder cancer, prostate cancer, or benign prostatic hyperplasia.

Gynecological cancer such as carcinoma or dysplasia of the cervix, cervical cancer, or cervical intraepithelial neoplasia is also possible.

ИЙО108| Skin cancers such as squamous cell carcinoma (SCC), epidermocytic carcinoma (SCC), malignant melanoma, HIV-induced Kaposi's sarcoma, and medial stage-type sarcoma are also possible.

Kaposi, basaloid follicular hamartoma, Bowen disease, cutaneous T-cell lymphoma, or sebaceous hyperplasia. Cancer can also be a pre-malignant or malignant skin growth.

It is also possible breast cancer such as metastatic breast cancer, colon cancer, endometrial cancer, eye tumor or head and neck cancer such as oral cavity cancer, pharyngeal cancer, lip carcinoma, nasal cavity or larynx cancer. (1) Combined treatment

ИЙО109| A method of treating a cancer or precancerous condition, a compound, or a composition disclosed herein may be combined or co-administered in antiblastoma therapy. This antiblastoma therapy can be the use of an antiblastoma drug, radiation therapy, surgery, or photodynamic therapy (PDT). (a) Anticancer treatment

ИЙО110| Cancer treatment may be radiation therapy such as X-rays, radiofrequency or microwave energy, ultrasound, radioisotope therapy, ionizing radiation, external beam or remote beam therapy, brachytherapy, or radiation therapy from a sealed or unsealed source. Anti-cancer treatment can also be surgery such as weight reduction, cryotherapy or surgical removal of tumors. (2) Photodynamic therapy 0111) PDT may involve irradiation with visible light of an aminoacridine-treated cell, which may also be treated with a photosensitizing agent. Light can have a specific wavelength and can activate the photosensitizing agent. An activated photosensitizing agent can generate singlet oxygen or a radical that can damage or kill a cell. The PDT may be as described in US Patents Mob,899,723 and Mob,693,093, the contents of which are incorporated herein by reference. r. Treatment of non-malignant manifestations 0112) Non-malignant manifestations in a patient suffering from them can be cured using the method. Treatment is indicated in cases of skin disorders such as psoriasis, common warts or hair loss. Ophthalmic disorders such as age-related macular degeneration, subfoveal choroidal vascularization, pathological myopia, reticuloendothelial cytomycosis, or choroidal neovascularization are also possible. Indications may also be such cardiovascular disorders as atherosclerotic plaque, intimal hyperplasia, atherosclerosis, or hemolytic lesions, or restenosis. with. Treatment of infection

ИО113| An infection in a patient suffering from it can be cured using the method. It can be a viral infection such as herpes, influenza, human immunodeficiency virus, or hepatitis A, B, and C.

It can also be a bacterial infection such as chlamydia, tuberculosis or mycoplasmosis. 01141 Infection can also be caused by eukaryotic parasites such as molds, giardia, toxoplasma or trypanosomes. Mold can be yeast. Trypanosomes can cause malaria and can be Plasmodium or Leishmania. and. Treatment of diseases associated with inflammation

ЙО115| Diseases or conditions associated with inflammation can also be treated using the method. Inflammation can be chronic, due to autoimmune disease, allergy, asthma, septic shock, graft-versus-host reaction, surgical wound, burn, or ischemic disorder. 4. Method of screening

ЙО116| Also provided herein is a method of screening compounds capable of modulating the adaptive heat shock response in a cell when such a compound is used in combination with inducing a heat shock response. This method may include inducing a heat shock response in a population of cells and contacting this population with a test compound. The number of dead cells from a population compared to a control can be an indicator of a compound's ability to modulate the adaptive heat shock response.

ИО117| Cells in the population may also contain an induced heat shock generator. In addition, the specified method may include measuring the concentration of the generator. This generator concentration compared to control may be an indicator of the compound's ability to modulate the adaptive heat shock response. The specified generator may contain such a copy of the NZR70O gene as NZR7OA1 or

NZR7OV. The specified generator may also contain the H5P70 protein. Said generator may also include a vector containing a heat shock response promoter operatively linked to the gene of the generator. This generator gene may encode a recognition marker, which may be luciferase, green fluorescent protein, yellow fluorescent protein, red fluorescent protein, or chloramphenicol acetyltransferase. 5. Composition 0118) This invention relates to a composition that may contain an inhibitor of the adaptive heat shock response. This composition may also contain a thermal shock reaction inducer. This composition may also contain a therapeutic agent, which may be an antiblastoma agent or a photosensitizing agent. This composition can be used in treatment by inducing a heat shock response in a cell treated with the composition, which can damage or kill the cell. and. Antiblastoma agent

I0O119| An antiblastoma agent can be any pharmacological agent or compound that induces apoptosis. A pharmacological agent can be, for example, a low molecular weight compound, a peptide, a polypeptide, a nucleic acid or an antibody.

IO120| The antiblastoma agent can be a cytotoxic agent or a cytostatic agent, or a combination thereof. Cytotoxic agents prevent the proliferation of cancer cells by (1) disrupting the ability of cells to replicate DNA and (2) inducing cell death and/or apoptosis of cancer cells.

Cytostatic agents act by modulating, interfering with, or inhibiting cellular signal transduction processes that regulate cell proliferation and, sometimes, at low plateau levels.

IO121| The cytotoxic agent can be an alkylating agent (including nitrogen mustard, ethyleneimine derivatives, alkylsulfonates, nitrosoamines or triazene), namely: uracil bitters, chlormethine, cyclophosphamide (Suiohapfb), ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, or temozolomide, antimetabolites (including folic acid antagonist, pyrimidine analog, purine analog, and adenosine deaminase inhibitor): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 56-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine; natural products or their derivatives (eg periwinkle alkaloid (Mipsa), antiblastoma antibiotics, enzymes, lymphokines, or epipodophyllotoxin): vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c, paclitaxel (TachoiF) , mithramycin, deoxycoformycin, mitomycin-c, 1-asparaginase, interferon (mainly IEM-a), etoposide or teniposide. The cytotoxic agent can also be navelbene, CRT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphatamide, ifosamide or droloxafine.

ИО122| A cytostatic agent can be an agent that affects microtubules and prevents cell mitosis. The agent affecting microtubules can be: allocolchicine (MZS 406042), halichondrin B (MZS 609395), colchicine (MS 757), colchicine derivatives (e.9., MS 33410), dolastatin 10 (MS 376128), maytansine (MZS 153858), risoxin (MZS 332598), paclitaxel (Taho, MZS 125973), Tahoko derivatives

(eg, M5C 608832), thiocolchicine (MC 361792), triphenylmethylcysteine (MZS 83265), vinblastine sulfate (M5C 49842), vincristine sulfate (MZS 67574), natural or synthetic epothilone such as epothilone A, epothilone B, or discodermolide (see begmise, (1996) Zsiepse, 274:2009), estramustine, nocodazole, MARA or the like. The microtubule-infecting agent may also be as described in the following articles: Viip5Ki (1997) U). Eat! Eat. 110:3055 3064; Rapaa (1997) Rhos. Mai). Asai. 5

BA 94:10560-10564; Mypyigaii (1997) Sapseg Nev. 57:3344-3346; Misoiaoi (1997) Maishte 387:268-272;

Mazda? (1997) Mine. Vioi SeiI. 8:973-985; Rapaa (1996) 9. VioI. Speth 271:29807-29812.

ИО123| A cytostatic agent can also be: epidophyllotoxin; antineoplastic enzyme; topoisomerase inhibitor; procarbazine; mitoxantrone; such a coordination complex compound of platinum, such as cis-platinum or carboplatinum; modifier of biological reactions; growth inhibitor; antihormonal therapeutic agent; leucovorin (folinic acid); tegafur; or hematopoietic growth factor. (01241 Cytostatic agent may be a hormone or steroid (including synthetic analogues) such as: 17-a-ethynylestradiol, diethistylbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisone, triamcinolone, chlortriazene, hydroxylprogesterone , aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, or zoladex.

ИО125)| The cytostatic agent can also be an anti-angiogenic agent such as a matrix metalloproteinase inhibitor, or another MESE inhibitor such as an anti-MESE antibody, or low molecular weight compounds such as 206474 and 506668. The cytostatic agent can also be a proprietary anti-Neg2 antibody

Sepepiesii, such an ESEK-inhibitor as EKV-569 (an irreversible inhibitor), or Itsiope antibody C225, which can be immunospecific for ESEBK, or a 5th-inhibitor.

ИО126)| A cytostatic agent can also be Sazodekhfb (bicalutamide, Aviga 7epesa), which can inhibit the proliferation of androgen-dependent carcinoma; the anti-estrogen Tatohiiep?, which can inhibit the proliferation or growth of estrogen-dependent breast cancer; or an inhibitor of cell proliferation signal transduction, such as an epidermal growth factor inhibitor, an inhibitor

Ne!-2, MEK-1-kinase inhibitor, MARK:-kinase inhibitor, RiZ-kinase inhibitor, 5-kinase inhibitor, or ROSPE inhibitor. Antiblastoma agents can also be the compounds listed in Table 1 with Sh.5.

Rgomysiope! Arrisaiop Mob1/102,913 dated October 6, 2008, the contents of which are incorporated herein by reference. r. Photosensitizing agent

I0127| A photosensitizing agent can be a compound that is absorbed by the cell and that upon exposure to light activates and causes the synthesis of a substance that can damage or kill the cell. The substance can be singlet oxygen or a radical. The photosensitizing agent can absorb light with wavelengths from 500 nm to 1.1 µM. The photosensitizing agent may be one of those disclosed in US Patent Nos. 6,693,093 and 7,018,395, the contents of which are incorporated herein by reference.

ИО128| For example, the photosensitizing agent can be green porphyrin; polypyrrole hydrophobic macrocyclic photosensitive compound; angelicin; lipofuscin; reaction center of photosystem II; BI-02-sui B-559 reaction center of photosystem II; chalcogen-pyrylium dye; chlorine; chlorophyll; coumarin; cyanine; such a DNA-related compound as adenosine, cytosine, 2'-deoxyguanosine-5'monophosphate, deoxyribonucleic acid, guanine, 4-thiouridine; 2'-thymidine-5'-monophosphate, thymidylyl(3'-5')-2'-deoxyadenosine, thymidylyl(3'-5')-2'-deoxyguanosine, thymine or uracil; a drug such as Adriamycin; afloqualone, amodiaquine dihydrochloride, chloroquine diphosphate, chiopromazine hydrochloride, daunomycin, daunomycinon, 5-iminodaunomycin, doxycycline, furosemide, hilvocarcin M, hilvocarcin M, hydroxychloroquine sulfate, lumidoxycycline, mefloquine hydrochloride, mequitazine, merbromine (mercurochrome), primaquine diphosphate, quinine sulfate, or tetracycline hydrochloride; flavin or a corresponding compound such as alloxazine, flavin mononucleotide, 3-hydroxyflavone, limichrome, limiflavin, b-methylalloxazine, 7-methylalloxazine, 8-methylalloxazine, 9-methylalloxazine, 1-methyllimichrome, methyl-2-methoxylbenzoate, 5-nitrosalicylic acid , proflavin or riboflavin; fullerene, metalloporphyrin; metallophthalocyanines; derivatives of methylene blue; naphthamide; Naphthalocyanine; such a natural compound as bis(4-hydroxyl-3-methoxylphenyl)-I, b-heptadiene-3,5-dione, 4-(4-hydroxyl-3-methoxylphenyl)-3-buten-2-one, M- formylkynurenine, kynurenic acid, kynurenine, 3-hydroxylkynurenine, BI-3-hydroxylkynurenine, sanguinarine, berberine, carmine, or 5;7;9(1);22-ergostatetrene-ZR-ol; derivatives of Nile blue;

M5ZAIIU (non-steroidal anti-inflammatory drug); aperilenquinone; phenol; pheophorbid; pheophytin; photosensitizing dimer or conjugate; phthalocyanine; porphycene; porphyrin; psoralen; purpurin; quinone; retinoid; rhodamine; thiophene; verdins; some vitamin; or xanthene dye (Keitopa and Satiip,

Rpotospet Biochemistry, 70(4):391-475 (1999)). with. Preparation of medicines

I0129| In addition to the above compositions, the compositions may also contain at least one such pharmaceutically acceptable ingredient as potassium alum, stabilizers, antimicrobial agents, buffer, dyes, flavors, adjuvants, etc.

0130) The composition can be in the form of tablets or cakes, which are formed by known methods.

For example, tablets and capsules for oral use may contain conventional excipients, including, but not limited to, binding agents, fillers, lubricants, disintegrants, and wetting agents. Non-limiting examples of binding agents are syrup, gum arabic, gelatin, sorbitol, tragacanth, starch paste, and polyvinylpyrrolidone. Non-limiting examples of excipients include lactose, sucrose, microcrystalline cellulose, corn starch, calcium phosphate, and sorbitol. Non-limiting examples of lubricants include magnesium stearate, stearic acid, talc, polyethylene glycol, and silicon dioxide. Non-limiting examples of disintegrants are potato starch and sodium starch glycolate. Non-limiting examples of humectants include sodium lauryl sulfate. Tablets may be coated by methods known in the art. 01311) The composition can also be liquid, non-limiting examples of which can be aqueous or oily suspensions, solutions, emulsions, syrups and elixirs. The composition may also be formulated as a dry product for reconstitution with water or another suitable vehicle prior to use. Such liquid preparations may contain additives, non-limiting examples of which are suspending agents, emulsifiers, non-aqueous media and preservatives. Non-limiting examples of suspending agents are sorbitol syrup, methylcellulose, glucose or sucrose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Non-limiting examples of emulsifiers include lecithin, sorbitol monooleate, and gum arabic. Non-limiting examples of non-aqueous media include edible oils, almond oil, fractionated coconut oil, fatty acid esters, propylene glycol, and ethyl alcohol. Non-limiting examples of preservatives are methyl or propyl para-hydroxybenzoate and sorbic acid.

I0132| The composition can also be prepared as suppositories, which can contain bases, non-limiting examples of which are coconut oil or glycerides. The composition can also be prepared as inhalation agents, non-limiting examples of which are a solution, suspension or emulsion, which can be administered as a dry powder or in the form of an aerosol using a propellant such as dichlorodifluoromethane or trichlorofluoromethane. The composition can also be prepared for transdermal administration in the form of preparations that contain aqueous or non-aqueous media and non-limiting examples of which are creams, ointments, lotions, pastes, medical plasters, overlays or films.

ЙО133| The composition can also be prepared for parenteral administration, non-limiting examples of which are injection or long-term infusion. Compositions for injection can be in the form of a suspension, solution or emulsion in an oily or aqueous environment and can contain technological ingredients, non-limiting examples of which are suspending agents, stabilizers and dispersants. The composition can also be prepared in the form of a powder for further mixing with a suitable medium, a non-limiting example of which is sterile pyrogen water.

I0134| The composition can also be prepared to form a depot of drugs by implantation or intramuscular injections. This composition can be created (for example, in the form of an emulsion in an acceptable oil) using such suitable polymeric or hydrophobic materials as ion exchange resins or as poorly soluble substances (for example, poorly soluble salts). 01351) The composition can also be prepared in the form of liposomal preparations. These liposomal preparations may contain liposomes that penetrate into the desired cells or into v5igaisht sogpeit, connect with cell membranes and thus deliver their contents into the cell. For example, liposomes such as those described in US patents Mo5,077,211, Mo4,621,023 and

No. 4,508,703, the contents of which are incorporated herein by reference. The composition intended for the treatment of skin damage can be administered before, during or after exposure of the skin of the mammary glands to ultraviolet radiation or the action of agents capable of causing oxidative damage.

Niosomes may serve as other suitable preparations. Niosomes are lipid formations that are similar to liposomes and have membranes consisting of a number of non-ionic fats, the shapes of which particles are effective for the transfer of compounds through the zighasht sogpeit. and. Dosage

ИО136| A therapeutically effective amount of the agent required for therapy will vary depending on the nature of the condition requiring treatment, the length of time the desired activity is required, and the age and health of the patient, and will be determined by the treating physician. The desired maximum dose is conveniently administered once, or divided into two, three, four or more subdoses per day, which are administered at appropriate intervals. Multiple dosing is often desirable or necessary.

I0137| When the composition is administered in combination with other therapeutic agents, its doses can be relatively reduced. In particular, the use of "targeting means" makes it possible to make the required doses relatively low. But some compositions can be administered in relatively high doses due to such (but not limiting) factors as low toxicity, high blood clearance, low tertiary amine cleavage rate. Accordingly, dosages of the composition can be from about 1 ng/kg to about 200 mg/kg, from about 1 μg/kg to about 100 mg/kg, or from about 1 mg/kg to about 50 mg/kg. Doses of the composition can be selected in the following, but not limiting intervals of approximately 1 μg/kg - 1 mg/kg, or 1 mg/kg -100 mg/kg. 0138) The proposed invention has many aspects, which are illustrated by the following non-limiting examples.

Example 1

Inhibition of the adaptive response to heat shock in combination with induction of this response can cause cell death

I0139| This example shows that the effectiveness of therapy aimed at heat shock can be significantly increased by the use of agents that block the induction of the adaptive heat shock response, that is, prevent the launch of the natural protective mechanism of inducing chaperones in cancer cells.

The adaptive response to heat shock can be selectively blocked by suppression of NZE-1, which is the main mediator of heat shock-induced transcription. Inhibitors of the adaptive heat shock response should be used in combination with heat shock-inducing compounds or factors that will increase their cytotoxicity. This cytotoxicity is more likely and pronounced for cancer, since the cancer cell is more dependent on chaperones than normal cells. 0140) This example also shows that the antimalarial drug acriquine (OC), its corresponding compound 9-aminoacridine (9AA) and other chemicals belonging to the carbazoles (C2) group act as non-genotoxic insertions in DNA, repressing the function of some classes of gene promoters, the induction of which is a consequence of the activation of signal transduction of metabolic pathways. Studies of the heat shock response of cells in the presence of these compounds show that they can suppress NZE-1-mediated transcription in a dose that is virtually independent of the method or agent used to induce the heat shock response.

ИО141|| Acrychin and 9AA were added to cultured cancer cells to test the effect of these compounds on the activation of p5p70 in response to treatment to induce a heat shock response. This heat shock response was induced by the use of known proteasome inhibitors MO132 and bortezomib,

РМАСИ17 (geldanamycin) as an inhibitor of H5POO and heat treatment (42-45 "C, 10-60 minutes). To track the heat shock reaction, the induced isoform of Ner/70 was detected by Western immunoblotting using an antibody that recognizes the induced form of the HOR70 protein, and Northern hybridization using a test preparation that specifically recognizes the mRNA of induced genes

NZR7OAT and NZR7OB'", but does not recognize the mRNA encoding the normally expressed NZC70 protein. OS and 9AA suppress the induction of the HOR7O protein (Fig. 1A). Acrychin and ZAA did not reduce the ability of MO132 or bortezomib to inhibit proteasome activity (Fig. 18), which suggests that the action of OS and 9AA is not associated with the inactivation of heat shock inducers.Moreover, induced duplicates

H5P7O accumulates in cells treated with heat shock inducers, but the presence of acrichin or 9AA blocks transcription (Fig. 2). Along with aminoacridines, a similar effect can be observed when the cell is treated with emetine, which is a nonspecific inhibitor of translation (Fig. 2). This confirms that the observed effect of SS and 9AA is a consequence of insufficient induction of heat shock proteins. 0142) Fig. 1 shows that acrichin (9C) and 9ZAA interfere with the synthesis of heat shock proteins

H5P?TO in a cell treated with proteasome inhibitors. Protein extracts from NeHa cells treated for 8 hours with 5 μM of the proteasome inhibitor MO132 alone or in combination with OS or 9AA were probed with antibodies to the induced form of NZR7O and to pyrin (gel loading assay) (Fig. TA). The results of IP myo proteasome activity of extracts from cells treated with MO132 alone or in combination with acriquine and 9AA show that OS and 9AA do not affect proteasome inhibition by MO132 (Fig. 18). 01431 Fig. 2 shows that OS and 9AA inhibit the transcription of the NZP70 gene in response to the action of proteasome inhibitors. 10 μg of total RNA from Her cells! and were treated for 5 hours with 0.1 μM bortezomib (Bgi7), or 5 mM MOo132 (MO) alone or in combination with 20 mM OS (Otsip), 20 μM 9AA, or 1 μM emetine (EM). The results were analyzed by Northern blotting with a test preparation specific for hybridization of NOR7OAIIV SZARON served as a control for RNA loading. Negr70(x) reflects induction of H5P 70 transcription. 01441 Fig. C shows that HOR70 synthesis induced by OMASI17 was sensitive to OS and 9AA. 10 μg of protein extracts from NeHa cells treated for 5 hours with 5 μM MOo132 (MO) or OMAS-17 alone or in combination with acrichin (Ocip) or 9AA were subjected to immunoblotting using antibodies to H5P70. A relative induction of HeP 70 (ipa) expression (by densitometry) was found.

ИО145| Fig. 4 shows that OS and 9AA inhibit the transcription of the H5P7OA1/B" gene induced by

RMACI17 and MO132 as determined by Northern hybridization of 10 μg total RNA from Nega cells treated with 1 μM OMAC17 for 5 h alone or in combination with 9AA. SARON was used as an RNA loading control. Neor7O(x) reflects induction of HOR70 transcription. 0146) Fig. 5 shows that the toxic effect of the combination of drugs on NeiYa cells, as shown by the results of the study of the formation of colonies by NeiYa cells, which were treated for 6 hours with the proteotoxic drug OMASI17 (1 μM) in combination with different concentrations of acrichin (10 or μM). Cells were collected, diluted 1:50, and examined for colony formation. Colony numbers were assessed after 7 days of growth using bromophenol blue for staining and 10 95 formaldehyde for fixation. The data shown are the average of three experiments. Inducers of heat shock and OS showed synergistic cytotoxicity.

ИО147| Fig. 6 shows the results of an experiment similar to the experiment illustrated in FIG. 5, but heat shock treatment (43"C, 30 minutes) was replaced by OMAS17. Synergistic cytotoxicity was found when OS was combined with heating.

IO1481 Fig. 7 shows that the sensitivity of cancer (that is, not normal) cells to heating increases under the influence of SOS and C (compound SVI 0137). CCC45 (renal epithelial carcinoma cells) and MKE-pTENT (normal renal epithelial cells immortalized by telomerase transduction) were suspended in culture medium, treated with different concentrations of OS or compound

SVI 0137 cells were then immediately heated in a water bath at 45 °C. The duration of incubation in a water bath is shown in different shades in the figure. After heating, the cells were placed in a 96-well plate and incubated for 24 hours in the presence of drugs, and then further incubated for 48 hours in a drug-free environment.Cell viability was examined using the Sei!Tag Wiene assay (AIatag

ViIse, Rgoted's firm). 0149) Fig. 8 shows that 5iRNA-mediated reduction of the nuclear complex component

EAST, 55RT1, and RAST kinases SKI! increased the sensitivity of cells to heat shock. HT1080 cells were transfected with 5iRNA pools generated against protein casein kinase II (PCII), 55RTT1 and (for control)

SARON (OPagtasop company) according to the manufacturer's protocol. After 24 hours, all cells were plated in a b-cell plate. After an additional 24 hours, the cells in the plate were heated in a water bath to 10°C for 10, 15, and 20 minutes. Cell viability was examined 48 hours after heating using the Seyi Tag Vicher test (Rgoteda).

ИЙО150| These results showed that the combined treatment of cancer cells with non-toxic or mildly toxic inducers of the heat shock response in the presence of inhibitors of NOE-1-mediated transcription in non-toxic or moderately toxic concentrations provides synergistic cytotoxicity, which is preferential for cancer compared to normal cells. The molecular mechanism of inhibition of NOE-1-mediated transcription by drugs OS, 9AA and C7 is associated with their ability to inhibit the activity of the nuclear chromatin-reconstructive complex EGAST, which is possibly essential for transcription controlled by U5E-1 and ME-KV (Fig. 8).

Example 2

Aminoacridines prevent HOR 70 activation in response to proteasome inhibition 0151) This example shows that aminoacridines prevent p5p70 activation in response to proteasome inhibition. Dysregulation of the induced forms of yp5p70 is a characteristic feature of the heat shock response to subsequent proteotoxic stress, which is a consequence of proteasome inhibition.

The effect of some antimalarial drugs on the synthesis of p5p70, activated by protease inhibition by the low molecular weight drug MO132, was investigated in cultured cells of Nei a. Emetine and acriquine (92) suppressed p5p70 synthesis in response to MO132 (Fig. 9A). 9-aminoacridine (9AA), which is substantially similar in structure to OS, has a similar inhibitory effect on H5p70 expression induced by MO132 (Fig. 98).

ИО152| Emetine is an inhibitor of gene translation in general; therefore, its ability to suppress induced synthesis of p5p70 is possibly a reflection of this property. However, OS and 9AA (as shown by the incorporation of labeled 355-methionine and 355-cysteine) do not affect protein synthesis in general at concentrations that are sufficient to completely suppress the induction of p5p7o0 (Fig. 9C). This indicates that compounds based on aminosacridine suppress the induced synthesis of p5p70 due to the action of another, more specific mechanism. Both OS and 9AA impaired the ability of MO132 to inhibit proteasome activity (Fig. 90). Thus, these drugs are not able to block p5p70 induction, only interfering with the development of proteotoxic stress due to proteasome inhibition.

ИО1531| The inhibitory effect of OS on the induction of p5p70 was recognized at the mRNA level, as shown by Northern hybridization of RNA from NeHa cells treated with MO 132 or another proteasome inhibitor - bortezomib - in the presence or absence of OS (Fig. 9UE). The hybridization test preparation used in this experiment was specific for the induced y5p7bAi mRNA and did not recognize the mRNA encoding the constitutive p5c70 protein. These data indicated that OS suppressed the accumulation of p5p/0 mRNA under the condition of proteasome inhibition. This effect was a consequence of transcriptional repression rather than stimulation of p5p70 mRNA degradation, because addition of OS after induction of NSC has no effect in the condition of p5p70 excess

mRNA. The inhibitory effect of OS and 9AA on gene expression was not universal. For example, 7psCig-induced activation of metallothionein gene transcription was not impaired by these compounds, provided that they did not reverse the induction of p5p70 mRNA (Fig. 9E). In addition, OS and 9AA both potently induce the transcription of target genes.

Example C

Akrichin blocks the induction of the heat shock response by inhibiting NOROO or hyperthermia

II0154| This example shows that acriquine blocks the heat shock response (HSR) induced by yp5po0 inhibition or hyperthermia. Functional inactivation of p5p0 by geldanamycin or its derivative 17-OMACE induces proteotoxic stress and activates NOC, including the synthesis of p5p70. As shown in Fig. In relation to proteotoxic stress induced by proteasome inhibitors, OS and 9AA also suppress p5p70 induction in response to p5poo inhibition, and this is seen at the transcriptional level (Figures 10A and B). Because OS and ZAA effectively repress 17-OMAS-activated transcription of the p5p70OAI gene, they do not affect the levels of several other (non-17-OMAS-induced) mRNAs, including the controls ZARON, rb5omeiA, and SCROA (Fig. 100).

ИО155| OS was also effective in blocking hyperthermia-induced NAC, as illustrated

Fig. 100. In this experiment, the activity of the transcription factor HER, which is recognized as essential for inducing the expression of p5p70 during H5E, was directly investigated. Cellular generators of H5RI were started by infecting cells with a lentiviral generator construct containing ESER cDNA under the control of a promoter sensitive to H5E. After treatment with bortezomib, 17-OMAS, or hyperthermia, H5RI-dependent ECER synthesis was induced as detected by fluorescence microscopy. Co-treatment of OS generating cells with one of the other three different inducers of proteotoxic stress blocks the accumulation of ESR. These results show that the antimalarial drug OS is a potent inhibitor of NSC.

Example 4

Akrichin inhibits the function of nuclear translocation of H5E1 and DNA binding

I0156| This example shows that acrichin inhibits the function of nuclear translocation of H5E1 and DNA binding. Activation of NZE1-mediated transcription requires the release of H5E1 from its complex with NZBROO in the cytoplasm. This occurs by the formation of homodimers and homotrimers of NZRI, which move into the nucleus and join specific recognition sites of NZRI within the promoters of genes induced during heat shock. The effect of OS and 9AA on this process was investigated to determine the point at which these compounds act to inhibit H5RI function. To evaluate intracellular localization and the ability of DNA to bind NOI complexes, an electrophoretic shear mobility test (hereafter EM5A) was performed using protein extracts purified from cytoplasm or cell nuclei and subjected to proteotoxic stress alone or in combination with OS or 9AA. The results of this experiment are shown in Fig. 11. Treatment of Nei cells with the drug MO132, heat shock, or the drug 17-OMAS led to specific activation

DNA in relation to H5RI binding both in cytoplasmic (Fig. 114A) and in nuclear extracts (Fig. 118).

OS and 9AA failed to reduce or modify the severity of these effects at any tested concentration (up to 100 mM) (data not shown and Fig. 118). These results showed that the inhibition of NORI-dependent transcription using OS and 9AA is observed below the point of its cytoplasmic activation, nuclear translocation and DNA binding.

I0157| The finding that OS and 9AA inhibit the nuclear translocation of H5BRI suggests that these compounds themselves were localized at least in part of the cell nuclei. This is confirmed by the results obtained using experimental modulation of their distribution inside the cell.

Bafilomycin as a lysosome-targeted antibiotic can markedly alter the intracellular distribution of lysosomotropic compounds, including acriquine and chloroquine, by inhibiting lysosomal IN--

ATP phases. Treatment of Ne cells with bafilomycin led to a change in intracellular localization

OS from predominantly lysosomal to predominantly nuclear (Fig. 11C), which was determined using a fluorescence microscope (because OS and 9AA are naturally fluorescent compounds).

The effect of bafilomycin treatment on the sensitivity of inducing y5p70 to OS was also investigated.

Bafilomycin increased the efficacy of OS as an H5K inhibitor in MO132-treated Hea cells, as shown in Fig. 110. In the presence of the indicated antibiotic, 5 mM OS inhibited the synthesis of p5r7o more effectively than 20 mM OS in its absence. These results revealed that the inhibitory activity of OS relative to H5BRI-mediated transcription depends on its concentration in the nuclei.

Example 5

Combination of acrichin with proteotoxic stress induces apoptosis

ИЙО158| This example shows that combining acrichin with proteotoxic stress induces apoptosis. The heat shock response (H5K) is adaptive for survival and can protect cells from various toxic factors. Rapid accumulation of induced chaperones such as p5p70 and p5p27 is indicative of prevention of cell death by heat shock or proteasome or p5p90 inhibitors. Therefore, the ability of OS and 9AA to suppress the induction of proteins encoded by H5REI genes can significantly increase the cytotoxicity of proteotoxic stresses. Nei a cells were treated with 17-OMAS or bortezomib alone or in combination with OS, and the effects of cell survival and induction of apoptosis were investigated. Figures 4A and 48, respectively, show that OS significantly increased the toxicity of 1 7-pMAS and bortezomib. The combined use of OS in concentrations that reduce cell survival by less than 5095 and practically non-toxic concentrations of 17-0OMAS or bortezomib causes a dramatic decrease in the number of cultured cells.

I0O159) It was also investigated whether the induction of apoptosis affects the number of living cells after drug treatment. Apoptosis was monitored by caspase-specific cleavage of keratin 18 and products

Cancer Consistent cell survival data show that combined treatment of OS and either bortezomib or 17-OMAS potently activated caspase-mediated protein cleavage that was barely detectable when the drugs were used alone (Figures 12C and 120). The caspase inhibitor 2MAYUO-EMK blocked this effect completely, which is confirmed by cases of caspase-specific proteolytic cleavage (Fig. 12E).

ИО160) To determine whether inhibition of p5p70 induction plays a critical role in mediated

OS of cell sensitization to bortezomib and 17-OMAS, the results of using OS and an alternative method of suppressing yp5p70 were compared. Nea cells were transfected with 5iRNA specifically designed against the induced form of Pp5p70. Transfected cells were unable to synthesize induced p5p70 after treatment with proteasome inhibitors or p5poo (Fig. 1203). Failure to synthesize pj5p70 under proteotoxic stress activated an apoptotic response similar to that induced by combined OS treatment and proteotoxic stress-inducing agents (Fig. 120).

The similarity of the biological effects of y5p70 depletion, mediated by 5iRNA and caused by OS treatment, relative to the sensitivity of cells to bortezomib and 17-OMAS suggests that the combination of OS toxicity and proteotoxic stress inducers is mediated by the suppression of NOK by OS acrichin.

Example 6

Antiblastoma effect of the combination of acriquine with the NB5POO inhibitor

ИО161| This example shows that combining acriquine with a p5ro0 inhibitor has antiblastoma effects. The finding that combining OS with proteotoxic stress-inducing drugs has a potent toxic effect on Neg/aip cells suggests that such combinations may have significant potential in anticancer therapy. To study the possibility of a therapeutic effect of the combination

OS/17-OMAS was tested for the development of tumors in two syngeneic mouse cancer models, namely: fibrosarcoma МСА205 and melanoma B-16. As an inducer of proteotoxic stress, 17-

OMAS, which has shown limited monotherapeutic antiblastoma efficacy. First, the effect of 17-OMACE and OS on the induction of H5K (Fig. 13A) and survival (Fig. 138) in cells was investigated

МСА20О5 and В-16 ip mio. Both cell lines responded to the drug similarly to NegGa cells, showing that these models can be reliably used to test the antiblastoma activity of the combination OS/17-

ROME. To confirm that these drugs similarly affect the induction of H5K in tumors growing i.p., MCA20O5 cells were implanted into C57VI/b mice, and the resulting tumors were treated with a single intratumoral injection of phosphate-buffered saline. (control), 25 mg of 17-OMAO alone and 1.25 mg of OS together with 25 μg of 17-OMAO. After four hours, the mice were killed and the RNA extracted from the isolated tumors was tested for induction of p5p70. As it shows

Fig. 13C, 17-OMAS treatment induced the synthesis of p5p/70 in tumors grown by IP, and OS prevented this induction. To study the effect of combinations of drugs on tumors grown by i.p. tumors, tumor-bearing mice МСА20О5 (Fig. 1303) and B-16 (Fig. 13E) underwent intratumoral injections of OS and 17-ОМАС separately or in combination. Tumor sizes after drug injections were measured regularly (every 2 days). In both experimental models, where OS and 17-OMAS were used alone, insignificant antiblastoma effects (in the inhibition of tumor growth) were found, while their combinations inhibited tumor growth. Tumor sizes were measured within 24 days after the injections. Tumors treated with injections of one drug began to outgrow tumors treated with injections of both drugs on the 16th day for B-16 cells and on the 18th day for MCA20O05 cells (figures 13A and 138). The use of the OS/17-OMAS combination not only prevented the growth of tumors, but also caused their regression, reducing the volume from -50 mm? to undefinable. These observations show that combining OS with inducers of proteotoxic stress induces apoptosis.

Example 7 Methods 1. Cell cultures, lentiviral vectors, 5iRNA transfection and drugs

ИО162| Neia cells were cultured in Juspresso's modified medium

Eadie5 (Ipiygodep/Zirso VK, Saghizrad, SA) with the addition of 10 95 fetal calf serum. Cells of mouse melanoma B16 and fibrosarcoma МСА205 were grown in complete medium KRMI-1640 with the addition of 2 mM I-glutamine, 100 μg/ml streptomycin, 100 units/ml penicillin, 50 μg/ml gentamicin (TesppoYodije5, Stgapa Iziapa, MU) . The expression of H5OE1-specific ESER was induced by introducing into RTK-tSMMU-ESER a lentiviral vector to the left of the minimal promoter of SMM (cytomegalovirus) with six copies of 5 СААСАСОТТСТАС 3 (5EO IO MO: 1) as regulatory elements

H5E1 (H5E). Cells were overnight infected with lentivirus MY 10 in the presence of 4 μg/ml polybrene.

The efficiency of infection was determined by the expression of ESER in response to heat shock at 43 "C for 60 minutes. From 70 to 90 95 cells were infected with the virus and expressed ESER in response to heat shock.

ИО163)| Her a cells were transfected with p5p70 5iRNA (Zapia Sty? Viotesppoiodu, Zapia Sty7, CA) for 24 hours to inhibit p5p70 expression after drug treatment. Control cells were transfected with siRNA-A, non-specific to p5p-70 (Zapia Stgy?7 Vioyesppoiodo, Zapia Sty7, SA). Cells were treated as described in the text with the proteasome inhibitors MO132 (SaiIriospet, Zap Oiedo, CA) and bortezomib (IS Iarogagogie5, UMorigp, MA), the p5re0 inhibitor 17-OMAa (IS Iarogayutievz, Movy,

MA), acriquine, emetine, quinine, chloroquine and ZAA (all from the company Zidt, her. GIocib5, MO).

Hyperthermia/heat shock was performed by incubation at 43 "C for 1 hour. 2. Western immunoblotting

I0O164| In KIRA-buffer (150 mM Mass, 1 95 sodium dodecyl sulfate, 10 mM Tgtgi5 (pH 8.0), 1 95 sodium deoxycholate, 1 95 MP-40), collective protein extracts from Nega cells containing a mixture of protease inhibitors ( Zidta, 5. Gotsi5, MO). Protein extracts were separated by electrophoresis in a polyacrylamide gel with a smooth concentration gradient of 4-20 95 with the addition of sodium dodecyl sulfate (Ipmigodep/Momekh, Sagizrai, SA) and then transferred to nylon-polyvinylidene fluoride membranes of RMOE (Ateg5ypat, Rizsaiauzhau, MU). Membranes were pre-incubated overnight in 5 95 milk solution and then incubated for 1 hour in 1 95 milk solution with 1:1000 solutions of primary antibodies. As primary antibodies, a rabbit antibody against induced forms of p5r7bAI (Azzau Yuezidp5/5ige55bep, Misia, VS, Sapada) and a rat apii-H5ORI (Azzau

Oezidp5/5igez55 (sep, Misiogia, VS, Sapada). Controls of protein loading were performed with rat antipyrin antibody (Ipziyshci 7ag Viospetieye deg I pdm/id-Mahitiyape-Opimeg:ytsa: Moipspep, Septapu) and rabbit antikeratin antibody K18 (Vygppat gezeagsp Ipbziyshe, SA). Apoptosis was detected by caspase-specific cleavage of K18 (Vygpyat gezeagsp Ipziyshe, CA) and RAKR (antibody from Zapia

are you Vioyesnpoiodu, Zapia Sty7, SA). HAP-conjugated secondary anti-rabbit, anti-rat, and anti-mouse antibodies were purchased from bZapia Sty7 Violyespoiod. JuOeiesiop Geadepi (ESI), which is a representative

Rezhip EiIteg (5peyop, ST). 3. Northern blotting and hybridization

ИЙО165| Total RNA (10 μg) from Neia, B-16, or MCA205 cells was examined by Northern blot hybridization with test preparations specific for the p5p7bAI, MT, re5KeiiA, SKRO4, and SARON genes.

The RSK fragment was generated from p5p7bAI cDNA (Ipmigodep, Sagizbride, CA) with primers specific for p5p7bAI, which encodes the sequence (AI5: 5 ССА ССА TOС ССА ССА dОаС АСА ССЗ (5EO I MO: 2);

Aa: 5 SAT BAA SSA TSS TST SSA SST 3)(5BO IO MO: 3). All remaining test preparations for hybridization were created by restriction digestion endonuclease from cDNA clones (OKISEME).

ИО166)| 355-protein-labeled Nega ip mimo cells were treated with drugs and their combinations according to the experimental protocol. Normal culture medium was replaced with methionine- and cysteine-free medium supplemented with 355-methionine and 55-cysteine (50 mCUR/ml) (Mem/ Epdiapa

Misieag/Regkip EIteg, 5peyop, ST), and the cells were incubated for an additional 60 minutes in the presence of drugs.

Cytoplasmic protein extracts were prepared according to the Oidtap protocol. Marked proteins were investigated by electrophoresis in a 10 95 polyacrylimide gel and autoradiography. 4. Testing the proteasome activity of migo

ИЙО167| To determine the effectiveness of the proteasome inhibitors MO132 and bortezomib alone and in combination with OS and 9AA, we treated Ne cells with the indicated drugs for 4 hours, purified cytoplasmic protein extracts and analyzed proteasome activity using fluorochrome proteasome substrate 1 (CaIBiospet, Zap Oiedo, CA) . 5. Electrophoretic shear mobility test (EM5A)

IO168| The activity of DNA in binding H5EI was investigated using EMZA. Briefly, cytoplasmic and nuclear protein extracts were purified according to the OIDTP protocol from control Nega cells and Nega cells treated with MO132 or 17-OMAS alone or in combination with

OS or 9AA. Next, 10 μg of each protein extract was analyzed using a C2-labeled double-stranded oligonucleotide (5 TtTos-ApO-TAS-AAS-STT-CTA-SAA-YaaST-TST-AcS 3)(5BEO IO MO: 4), specific for the H5E1 tying. For comparison, a 50x excess of unlabeled oligonucleotides was added to the protein extracts together with 3P-labeled double-stranded oligonucleotides. The protein extracts were pre-incubated with NZE1-specific antibodies (A5zzau Oevzidp5/5igez5bep, Misiopa, VS, Sapada) for 15 minutes at room temperature before by adding a 3P-labeled double-stranded oligonucleotide to perform EM5A. 6. Cell viability testing

IO169| A cell survival test was used to determine the cytotoxicity of drugs and their combinations. Cells were grown to confluence of 75-80 95 and then treated for 4 hours with different compositions of 1 mM 17-OMAS, 0.1 mM bortezomib and 10 mM or 20 mM OS. Cells were trypsinized, diluted 1:50 and plated in a b-cell plate. Cell survival was determined 72 hours after staining with methylene blue and fixation with 10 95 formaldehyde. Methylene blue was extracted with a 0.1M solution of NS, and its spectral absorbance was measured at 560 nm. 7. IP testing of tumor development in mice

0170) MCA205 and B-16 cells were detached from plastic Petri dishes using a 0.5% solution of trypsin and ethylenediaminetetraacetic acid, washed and resuspended in sterile phosphate-salt buffer solutions. These cells were injected into mice of the C57VI/6 line transdermally in the exposed abdominal area (3 x 109 cells in one injection per mouse). Tumors were measured in two directions and their volume was calculated according to the formula: M mm" - tumor length x (tumor width). On the eighth day after tumor implantation, the typical volume was "50 mm3. Tumor-bearing mice were divided into four groups of five animals each were subjected to intratumoral drug injections from day 9. The control group received injections of only phosphate-salt buffer solutions on days 9, 10, 11, and 12. Mice in group 2 received other injections of 1.25 mg of OS in phosphate-salt buffer solutions on days 9 and 12. Mice in group C received injections of 25 μg of 17-OMAS in phosphate-salt buffer solutions on days 9, 10, 11, and 12. Mice in group C 4 received injections of 1.25 mg of OS-25 μg of 7-OMAS on days 9 and 10 and 25 μg of 17-OMAS on days 11 and 12. OS was injected only twice, because this drug is stable inside the cells. It can be detected in cells 72 hours after injection and medium replacement Tumors were measured every two days after drug injections for 24 days Animals with tumor volumes of 500 mm3 and more were euthanized.

Example 8

The combination of an ineffective dose of acrichin and local hyperthermia provides antiblastoma effect in a mouse model

ЙО171| This example shows that combining an ineffective dose of acrichin with local hyperthermia shows an antiblastoma effect in mouse model experiments. Model tumors from C26 mouse colon carcinoma cells were formed and developed in syngeneic mice of the BaIB/s line. When the tumors reached a volume of 50-150 mm3, mice were treated orally with acriquine (500 mg/kg), or local hyperthermia (1 hour at 41.5 "С) or both (acryquine was administered 2 hours before hyperthermia). After five days of tumors were measured, and these results are shown in Fig. 14 with calculations of the statistical probability of the observed differences.

Example 9

Carbazole prevents the expression of HOR 70 in response to proteotoxic stress (hereafter PS)

I0172| This example shows that carbazole can inhibit the expression of p5p70 in response to PS.

Since aminoacridines (AA) effectively inhibit the expression of p5p70 in response to a variety of PS inducers, the specific activity of these compounds is not very significant. They are active in micromolar concentrations. In order to find reagents more effective than AA, compounds with a structure similar to AA were investigated. In order to test the ability of carbazoles (SEQ2) to reduce the expression of p5p70 in response to PS, cells were treated with Mo132 and different amounts of several SCVAs, followed by analysis of p5p70 expression by Northern blotting. SCVA Me159 and Me198 showed activity in this experiment. The selected compounds Me159 and Me198 were active starting at a concentration of 1 µM (Fig. 153. SLE Mo198 was an effective inhibitor of NZE1-specific expression of CEP in cells treated with bortezomib (Fig. 16), and SLE; Mo159 and Mo198 were active in inhibiting the expression of the SER generator in response to hyperthermia (Fig. 17).Thus, SLE can increase PS toxicity with a better therapeutic index than AA.

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Fig. 17

Claims (15)

1. The method and induction of cell death, consisting of: (a) inhibition of the adaptive response to heat shock in the cell; and (0) inducing a heat shock response in the cell, wherein the inhibitor of the heat shock response is an aminoacridine or a carbazole selected from the group consisting of SVI0137, SVI0197, SVI.0198, SVI.0100, SVI.0159, SVI.0212, SVI,0174 1 SVI.0175. 2. The method according to item I, in which the heat shock reaction is induced by introducing a heat shock inducer into the cell. 3. The method according to claim 2, in which the heat shock inducer is geldanamycin, a proteasome inhibitor, arsenite or ethanol. 4. The method according to claim 3, in which the proteasome inhibitor is bortezomib. 5. The method according to item I, in which the heat shock reaction is induced by increasing the internal temperature of the cell. b. The method according to claim 5, in which the temperature of the cell is increased using a heating agent. 7. The method according to claim 1, in which the aminoacridine is selected from the group consisting of acriquine and 9-aminoacridine. 8. The method according to claim 1, in which the cell is a cancer cell. 9. The method according to claim 8, in which the cancer is selected from the group consisting of metastatic breast cancer, bladder cancer, lung carcinoma, esophageal cancer, basal cell carcinoma, malignant melanoma, eye tumor, head and neck cancer. 10. The method according to claim 1, in which the second treatment is associated with inducing a heat shock reaction in the cell. 11. The method according to claim 10, in which an antiblastoma agent is used in the second treatment. 12. The method according to claim 6, in which the temperature is increased to at least 39-60 C. 13. The method according to op. 6, in which the temperature is increased by infrared radiation. 14. The method according to claim 13, in which radiation with a wavelength of 25, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.68, 5, 9,6,6,1, 6,2, 6,3, 6,4, 6,5, 6,6, 6,7, 6,6, 6,9, 7, 7,1, 72,73, 7,A,7,5,7,6,7,7,7,6,7,9, 8, 8,1, 8,2, 8,3, 8,4, 8,5, 8,6, 8.7, 5.8, 8.9.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11, 3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12, 6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13, 9, 14, 141, 14.2, 143, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9 or 15 µm. 15. The method according to claim 6, in which the temperature is increased by a heat source selected from the group consisting of: an electroluminescent device, a laser diode, an emission laser with a vertical resonator surface, an LED, and a resistive incandescent lamp.