JP2021155396A - Prevention and/or treatment agent for preventing and/or treating adult t cell leukemia - Google Patents

Abstract

To provide a treatment agent having a novel action mechanism with respect to ATL and HTLV-1 related disease.SOLUTION: Inventors of the invention have found a fact that, an HIV integrase inhibitor MK-2048 promotes activity of an endoplasmic reticulum stress (PERK path) selectively, with respect to HTLV-1 infected cells, and induces apoptosis. On the basis of the result, using MK-2048 of the invention, the inventors have found that, a treatment agent having a novel action mechanism with respect to ATL and HTLV-1 related disease, can be provided. In addition, the MK-2048 of the invention is a novel endoplasmic reticulum stress targeting therapy agent with respect to ATL, so that, it is possible to provide a combined treatment agent with an anti HTLV-1 agent having other action mechanism, by expectation of a synergic effect.SELECTED DRAWING: None

Description

The present invention relates to diseases derived from infection with human T-cell leukemia virus type 1 (HTLV-1) (adult T-cell leukemia (ATL), HTLV-1-related myelopathy and HTLV-1 vaginitis). ) New preventive and / or therapeutic agents. In particular, the HIV integrase inhibitor MK-2048 was used to induce HTLV-1 infected peripheral blood mononuclear cells (PBMCs) to undergo vesicle (ER) stress-mediated apoptosis and infect HTLV-1. By selectively killing and eliminating the PBMCs, the onset of HTLV-1-related diseases is prevented and / or treated.

HTLV-1 (Human T-cell leukemia virus type 1), which is a type of retrovirus, is known to infect T cells and cause adult leukemia (ATL) and HTLV-1-related myelopathy (HAM / TSP). ing. HTLV-1 virus carriers exist mainly in southwestern Japan such as Kyushu and Okinawa, and it is said that there are about 1.08 million people nationwide. It is believed that about 5% of ATLs develop. However, the prognosis of the onset ATL is very poor compared to other leukemias, and the 5-year survival rate is said to be 12 to 17%.
Regarding HTLV-1, research on therapeutic agents has been delayed due to the severe regional bias among patients and the low incidence rate, but the number of people infected with HTLV-1 and the post-onset of related diseases Due to the severity of its symptoms, it is one of the viruses that is currently receiving a great deal of attention.

Mogamulizumab (anti-CC chemokine receptor 4 humanized monoclonal antibody) was launched in February 2012 as a therapeutic agent for ATL and HTLV-1-related diseases, and the immunomodulator lenalidomide was launched in March 2017 (Non-Patent Document). 1). Although mogamulizumab has been shown to be effective against CCR4-positive adult T-cell leukemia and lymphoma, these new drugs have shown some effect on HTLV-1-related diseases, but are effective in improving disease progression. It is low and some serious side effects have been reported (Non-Patent Document 2).
Therefore, new, safe and effective treatments are still in demand for HTLV-1-related diseases. As one of the attempts, a therapeutic agent for HIV (human immunodeficiency virus) belonging to the same retrovirus family has been diverted (Non-Patent Documents 3 to 4). However, a drug with a good balance between therapeutic effects and side effects has not yet been found.

Package insert "Rebramid Capsule" Celgene (additional efficacy in March 2017) Pharmaceutical Interview Form "Poterigio Intravenous Infusion 20 mg" Kyowa Kirin (Revised in July 2019) Rabaaoui S.A. et al, Antimiclob Agents Chemother. 2008; 52 (10): 3532-3541. Aluminum MEet al, Antimiclob Agents Chemother. 2011; 55 (5): 2011-2017.

An object of the present invention is to provide a compound that selectively kills and eliminates HTLV-1-infected T cells in order to develop a prophylactic and / or therapeutic agent for the onset of HTLV-1-related diseases.

In order to develop a therapeutic agent for HTLV-1-related diseases, the present inventors used a therapeutic agent for HIV (human immunodeficiency virus) belonging to the same retrovirus family as HTLV-1, and examined its effect. To that end, BMS-707035 (BMS), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), cabotegravir (CAB), and MK-2048, which are known as HIV integrase inhibitors, were used. .. The HIV integrase inhibitors of FIG. 1 are HTLV-1-infected cell lines (KK1, ED, SLB-1, C8166), HTLV-1 non-infected T-cell leukemia cell lines (Jurkat and MALT-4), and PBMC of healthy subjects. We investigated what kind of cell proliferation inhibitory effect it has on (peripheral blood mononuclear cells). As a result, it was shown that the HIV therapeutic agent of FIG. 1 has different effects on the HTLV-1-infected cell line as shown in FIG. 2 due to the difference in chemical structure. It was revealed that MK-2048 represented by the following formula (1) exhibits a specific growth inhibitory effect particularly on HTLV-1-infected cell lines.

When the present inventors further investigated the mechanism of action of MK-2048 on HTLV-1-infected cell lines, MK-2048 was found to be endoplasmic reticulum (ER) on HTLV-1-infected T cells, as shown in FIG. It was revealed that the gene expression of the stress response was increased. In particular, it has been clarified that it induces an increased expression of an apoptosis-promoting gene (DNA damage-induced transcript 3 protein: DDIT3) related to endoplasmic reticulum stress. This apoptosis-promoting gene is known as C / EBP-like protein (CHOP).
However, in HTLV-1 non-infected T cells, MK-2048 does not cause increased expression of endoplasmic reticulum stress-related apoptosis-promoting genes. That is, MK-2048 selectively induces activation of the PERK pathway of endoplasmic reticulum stress on HTLV-1-infected T cells and induces apoptosis associated with endoplasmic reticulum stress on HTLV-1-infected T cells. It became clear.
Among the HIV integrase inhibitors in the present invention, the present inventors specifically induce endoplasmic reticulum stress-related apoptosis of MK-2048 to selectively kill and eliminate HTLV-1-infected T cells. The new mechanism of action of MK-2048 was clarified. The present inventors have completed the present invention based on these findings.

That is, the gist of the present invention is as follows.
(1) An endoplasmic reticulum stress promoter for HTLV-1-infected peripheral blood mononuclear cells (PBMC) containing MK-2048 as an active ingredient.
(2) The endoplasmic reticulum stress promoter according to (1) above, wherein the PBMC is a T cell.
(3) The endoplasmic reticulum stress promoter according to (1) or (2) above, wherein the HTLV-1-infected PBMC is CD4 ⁺ CADM1 ^{+ cells.}
(4) The endoplasmic reticulum stress promoter according to any one of (1) to (3) above, wherein the promotion of endoplasmic reticulum stress is promotion of gene expression in the PERK pathway.
(5) The endoplasmic reticulum stress promoter according to (4) above, wherein the gene is CHAC1, TRIB3 or CHOP / DDIT3.
(6) The endoplasmic reticulum stress promoter according to (4) or (5) above, wherein the gene is CHOP / DDIT3.

(7) An apoptosis-promoting agent for HTLV-1-infected PBMC containing MK-2048 as an active ingredient.
(8) The apoptosis-promoting agent according to (7) above, wherein the PBMC is a T cell.
(9) The apoptosis-promoting agent according to (7) or (8) above, wherein the HTLV-1-infected PBMC is CD4 ⁺ CADM1 ^{+ cells.}
(10) The apoptosis-promoting agent according to (9) above, wherein the promotion of apoptosis is a decrease in gene expression of GRP78 and an increase in gene expression of CHAC1, TRIB3 or CHOP / DDIT3.
(11) The apoptosis-promoting agent according to (10) above, wherein the enhancement of the PERK pathway is an enhancement of CHOP / DDIT3 gene expression.

(12) A selective exclusion agent for HTLV-1-infected PBMC containing MK-2048 as an active ingredient.
(13) The selective exclusion agent according to (12) above, wherein the PBMC is a T cell.
(14) The selective exclusion agent according to (12) or (13) above, wherein the HTLV-1-infected PBMC is CD4 ⁺ CADM1 ^{+ cells.}
(15) A prophylactic or therapeutic agent for adult T-cell leukemia (ATL) and / or HTLV-1-related diseases containing MK-2048 as an active ingredient.
(16) The prophylactic or therapeutic agent according to (15) above, wherein the prevention or treatment is the selective elimination of HTLV-1-infected PBMC.

Although MK-2048 of the present invention is an HIV integrase inhibitor, it strongly suppresses cell proliferation against HTLV-1-infected T cells. In the present invention, it is clarified that MK-2048 increases gene expression of endoplasmic reticulum stress (PERK pathway) in HTLV-1-infected T cells and induces apoptosis associated with endoplasmic reticulum stress as its mechanism of action. I made it. In addition, since the expression of GRP78, which is a dominant factor of endoplasmic reticulum stress, is reduced in HTLV-1-infected T cells, HTLV-1-infected T cells are MK-2048-induced apoptosis associated with endoplasmic reticulum stress (cells). It was found to be highly sensitive to death).
From these facts, it was found that MK-2048 can be used to induce apoptosis associated with endoplasmic reticulum stress in HTLV-1-infected T cells and selectively kill and eliminate HTLV-1-infected T cells. These findings have made it possible to provide MK-2048 as a useful prophylactic and therapeutic agent for ATL and HTLV-1-related diseases.

The structural formulas of the compounds of the HIV integrase inhibitors evaluated in the present invention (BMS-707035 (BMS), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), cabotegravir (CAB), and MK-2048). It is a posted figure. It is a figure showing the growth inhibitory effect of HTLV-1-infected T cells by an HIV integrase inhibitor. It represents the viability of the indicated cells after treatment with an HIV integrase inhibitor (50 or 100 μM). The survival rate is expressed by measuring the viable cells with and without the HIV integrase inhibitor with CellTiter and expressing the relative ratio. Numerical values were expressed by the average value and standard deviation from three experiments. It is a figure which showed the situation which MK-2048 increased the expression of the gene related to endoplasmic reticulum stress response (UPR) in HTLV-1-infected T cells. Using three types of HTLV-1-infected T cells, a microarray data disruption tool Ver. It is a figure created using 3.2. The color intensity represents the number of cell types in which gene expression has fluctuated. CHAC1 shown in yellow is a novel apoptosis-promoting factor for endoplasmic reticulum stress, and the gene expression was most elevated in all three types of HTLV-1-infected T cells used in this analysis. It is a figure showing that MK-2048 (0 to 50 μM) strongly inhibits the growth of HTLV-1-infected cell lines (SLB-1, MT-2, MT-4, C8166) in a dose-dependent manner. Cell viability was measured with CellTiter and shown as a relative ratio to control (DMSO added). It is a figure which showed the action of MK-2048 on the HTLV-1-infected cell line and the non-infected cell line. The IC50 value (μmol / L) was treated with MK-2048 for 72 hours, and HTLV-1-infected cell lines and non-infected cell lines were evaluated. Compared with HTLV-1 non-infected leukemia cell lines (Jurkat and MOLT-4), HTLV-1-infected cell lines (SLB-1, MT-2, MT-4 and C8166) had lower IC50 values for MK-2048. , MK-2048 show a selective growth inhibitory effect on HTLV-1-infected cell lines. It is a figure which showed that MK-2048 induces the activation of caspase 3/7 and the decrease of the cell proliferation rate in the HTLV-1-infected cell line. In FIG. 6 (A), treatment with MK-2048 (0 to 50 μM) for 24 hours shows the activity of caspase 3/7 in the indicated cells. The activity value of caspase 3/7 in MK-2048 treated cells was shown as a relative ratio to MK-2048 untreated cells. In FIG. 6B, the cell proliferation rate was measured at the same time. The proliferation rate of MK-2048 treated cells was measured with a cell titer and expressed as a relative ratio to MK-2048 untreated cells. It is a figure which showed that MK-2048 increased the expression of the gene associated with the endoplasmic reticulum stress response in HTLV-1-infected cells. In FIG. 7A, the cells were cultured for 16 hours with and without MK-2048 treatment (25 μM), and the changes in gene expression with respect to the indicated cells were expressed as relative ratios. About 20000 genes were examined. Red dots represent genes whose expression has been statistically significantly varied (P-value <0.01, fold change (FC)> 2). Blue dots are genes associated with the significantly altered endoplasmic reticulum stress response. Gene expression did not change for cell line OATL4. In FIG. 7B, the result of the microarray is represented by a Venn diagram. The number of genes whose expression was significantly altered in the displayed cells by MK-2048 treatment is shown in the overlapping circles. Blue circles represent SLB-1 cells, red circles represent ED cells, and green circles represent KK1 cells. Genes related to endoplasmic reticulum stress response are listed in the table in the figure. In KK1, SLB-1 and ED cells treated with MK-2048, the number of genes whose expression was significantly changed was 215, 362 and 33, and among the genes whose expression was significantly changed, 19 genes were 3 types. It was common to all cell lines. It is a figure which showed that MK-2048 induces endoplasmic reticulum stress-involved apoptosis in MK-2048-sensitive HTLV-1-infected cell lines (SLB-1, MT-2, KK1, and ED). The cells were cultured for 16 hours with and without the addition of 25 μM MK-2048, and the expression level of CHOP mRNA in the indicated cells was evaluated. The mRNA expression level shown in the figure was corrected by GAPDH, compared with the mRNA expression level in control (DMSO-added) cells, and expressed as a relative value. It is a figure which showed the influence on the drug efficacy of MK-2048 by the inhibition of PERK pathway activation. The changes in cell viability, caspase-3 / 7 activity and mRNA expression levels of CHAC1 and CHOP with and without addition of MK-2048 and PERK pathway activation inhibitor (GSK2606414) were shown. The numerical values in the figure represent the relative ratio with and without the addition of MK-2048. It is a figure showing that the mRNA expression level of GRP78 is increased in MK-2048 resistant HTLV-1-infected T cells. The mRNA expression level of GRP78 in the GAPDH-corrected cells was compared with the mRNA expression level in Jurkat cells and expressed as a relative ratio. It is a figure showing that the gene expression of GRP78 is suppressed in the HTLV-1-infected cell. Asymptomatic HTLV-1-infected PBMC (9 samples) was used as HTLV-1-infected cells to evaluate the expression level of GRP78 mRNA. In the left view of FIG. 11, the flow of the HTLV-1 infected individuals PBMC asymptomatic sites obtained by separating by cytometry ^CD4 + CADM1 ^- (HTLV-1 uninfected) cell populations and ^{CD4 +} CADM1 + (HTLV-1 In the infected) cell population, the gene expression level of GRP78 was expressed as a relative ratio. In the right figure of FIG. 11, the bar graph of the left figure is represented as a point graph. It is a figure of the box-shaped plot showing the mRNA expression level of GRP78 in PBMC of ATL patients (smoldering type (asymptomatic) 3 persons, chronic 20 persons, acute 26 persons). ^{Individual CD4 +} cells (21 persons) of healthy subjects were used as controls. Gene expression levels were assessed from datasets stored on the NCBI GEO website. It is a figure showing that MK-2048 significantly reduces the amount of provirus in HTLV-1-infected cells of asymptomatic HTLV-1-infected persons. PBMCs (# 1-4) from 4 asymptomatic infected individuals are cultured at a given concentration of MK-2048 for 4 days. In (A) of the figure, the amount of provirus (number of copies / 100 cells) of the cells after culturing was measured by quantitative PCR. The bar graph represents the average value of the relative ratio of the proviral load of the MK-2048 treated cells to the MK-2048 untreated cells. (B) in the figure represents the number of cells measured at the same time. The bar graph represents the average value of the relative ratio of the number of cells of MK-2048 treated cells to that of MK-2048 untreated cells. It is a figure which showed that MK-2048 induces endoplasmic reticulum stress apoptosis specifically in HTLV-1-infected cells. PBMCs were isolated from the peripheral blood of asymptomatic HTLV-1 infected individuals and treated with 25 μM MK-2048. (A) is dyed separately with the indicated antibody after treatment with 25 μM MK-2048 for 24 hours, and shows that CHOP is expressed only in ^{infected cells (CD4 + CADM1 +).} (B) is a representative FACS profile of CADM1 and CD4 in ^{a DAPI-} CD14 ^- cell population after 16 hours of treatment with 25 μM MK-2048 (left) and expression of anexin V in the ^DAPI- CD14 ^- CD4 ^{+ cell fraction.} the amount of infected cell population ^(CD4 + CADM1 ⁺⁾ and non-infected cell population ^(CD4 + CADM1 ^-) is shown in comparison with (right). In (C), infected cell population percentage of annexin ^{V +} cells ^{(CD4 +} CADM1 +) and non-infected cell populations ^- indicated by box chart summarizes the analysis results of the comparison to 6 persons with ^(CD4 + CADM1) There is.

The "HTLV-1-infected peripheral blood mononuclear cell" of the present invention refers to a mononuclear cell (mononuclear cell) containing monocytes and lymphocytes present in the peripheral blood of an HTLV-1 virus-infected person. In the present invention, it particularly refers to lymphocytes infected with HTLV-1 virus. The HTLV-1 virus has no subjective symptoms when infected, and once infected, it survives in lymphocytes and causes serious illness in a small number of infected people. Adult T-cell leukemia (ATL), which is one of the diseases, is leukemia (including lymphoma) caused by tumorigenesis of a type of lymphocytes (T cells) infected with HTLV-1.
As for the diagnosis of ATL, for example, a patient having more than 5% of abnormal lymphocytes (HTLV-1-infected cells) in peripheral blood can be diagnosed as ATL. In addition, since ATL is a disease caused by the monoclonal proliferation of T cells in which HTLV-1 is integrated into the genome as a provirus, the diagnosis of ATL is that of ATL cells in a blood sample obtained from a subject. It can be diagnosed by detecting that HTLV-1 provirus is monoclonally incorporated into DNA by Southern blot method, polymerase chain reaction (PCR), or the like (Re-Table 2018-25978). In addition, as a test method for diagnosing that the subject is an HTLV-1 carrier, a particle agglutination method (PA method), a chemiluminescence method, an indirect immunofluorescence method using a virus-infected cell as an antigen, and a Western blot are used. The method and the like are known and can be used as appropriate. In Western blotting, in the sample obtained from the subject, the antibody against the viral envelope protein (gp46) was positive, and one or more of the antibodies against the three core proteins (p19, p24 and p53) were positive. In some cases, the subject can be diagnosed as an HTLV-1 carrier. Alternatively, whether or not the subject is a carrier may be diagnosed by a polymerase chain reaction (PCR) that supplementarily amplifies the provirus genome of HTLV-1.
HTLV-1 virus-infected T cells can be detected as CD4 and CADM1 (Cell Attachment Molecular 1) -positive cells by immunostaining or flow cytometry. In addition, their clonality is investigated using the repertoire diversity of the T cell receptor (TCR), the genome integration position of the HTLV-1 provirus, etc., and if it is monoclonal, the detected cells are identified as ATL cells. be able to.

The "endoplasmic reticulum stress promoter" of the present invention refers to an agent that promotes or enhances endoplasmic reticulum stress of cells and induces cells to undergo apoptosis. In cells, the endoplasmic reticulum (ER) environment is affected by internal and external stress (eg, viral infections, nutrient deficiencies (amino acids, sugars, lipids), ischemia, heat shock, oxidative stress, etc.) at the cellular level. It is disturbed, resulting in a condition in which protein folding in the ER is impaired (endoplasmic reticulum stress). When cells are exposed to endoplasmic reticulum stress, it is known that the endoplasmic reticulum stress response (UPR) shown in FIG. 3 controls the endoplasmic reticulum function in response to the environmental change to adapt to the stress. The endoplasmic reticulum stress response can be broadly divided into three routes as its activation mechanism. Each endoplasmic reticulum stress transmitting protein (IREl, ATF6, PERK) senses the accumulation of unfolded protein in the endoplasmic reticulum. That information is transmitted to the cytosol and nucleus to activate the endoplasmic reticulum stress response (UPR). When an incompletely folded protein that exceeds the protein folding ability in the endoplasmic reticulum is produced, a response that reduces the load on the endoplasmic reticulum occurs by first stopping the synthesis of the protein (translation suppression). This is controlled by phosphorylation of eIF2α (eucaryotic initiation factor 2α subunit) required for translation initiation by PERK. Next, in order to increase the protein folding ability in the endoplasmic reticulum, a molecular chaperone that performs protein folding is induced. This is mainly controlled by activating ATF (Activating transcription factor) 6 by being cleaved by ATF6 (N). In addition, endoplasmic reticulum-related degradation (ERAD), which is withdrawn from the endoplasmic reticulum into the cytosol and degraded by the ubiquitin proteasome system, is promoted in order to remove the unfolded protein accumulated in the endoplasmic reticulum. This is mainly controlled by IRE1 (Inositol-requiring enzyme1) selectively splicing XBP1 (X-box-binding protein 1) into an active transcription factor. In cells with a disorder in which endoplasmic reticulum stress cannot be relieved by such an adaptive response, phosphorylated eIF2α induces the transcription factor AFT4 (activating transcription factor 4), and AFT4 is also an apoptosis-promoting transcription factor CHOP (C). / EBP homogous protein) is eliminated by apoptosis by inducing expression or the like. That is, the present invention refers to a drug that promotes or enhances endoplasmic reticulum stress in cells, induces endoplasmic reticulum stress into a state in which it cannot be eliminated, and induces apoptosis in cells.

The "CHOP / DDIT3" of the present invention represents a protein represented as CHOP or DDIT3, and also refers to a gene corresponding to the protein. CHOP, as shown in FIG. 3, is a protein that is significantly induced by upregulation of AFT4 in the endoplasmic reticulum stress PERK pathway and is activated in response to misfolded and unfolded proteins. Activation of CHOP induces apoptosis. Therefore, CHOP is considered to be a pro-apoptotic marker of endoplasmic reticulum stress-dependent cell death. CHOP is known to form heterodimers with transcription factors C / EBP and LAP, act dominantly negatively to inhibit them, and induce endoplasmic reticulum stress-dependent apoptosis.
The "CHAC1" of the present invention represents a protein represented as a cation transport regulator homolog 1 (CHAC1), and refers to a gene corresponding to the protein. It has been reported that the CHAC1 protein has a γ-glutamyl cyclotransphase activity and has a function of degrading glutathione, which is a major substance involved in the maintenance of the intracellular redox state. Recently, it has been reported that CHAC1 is a novel apoptosis-promoting factor induced by endoplasmic reticulum stress (Mungrue IN et al, J. Immunol 182,466-476 (2009)). In addition, among the three major endoplasmic reticulum stress response pathways, the PERK pathway has been suggested to be involved in the expression of the CHAC1 gene (Romanoski CE et al, Circ Res 109, e27-41 (2011)). It has also been reported that the expression of CHAC1 mRNA is increased when treated with a endoplasmic reticulum stress inducer such as tunicamycin or tabsigargin, and it is considered to be a endoplasmic reticulum stress-inducing gene. It has been clarified that ATF4 is directly involved in the expression of CHAC1. Furthermore, it is known that CHAC1 has increased mRNA expression when the prognosis of breast cancer or ovarian cancer is poor, and that knockdown of CHAC1 expression in breast cancer or ovarian cancer cells suppresses cell migration. It is also known to be associated with cancer (Goebel Get al, Br J Cancer 106, 189-198 (2012)).

The "TRIB3" of the present invention refers to a protein represented as a human homologue (Tribbles-related protein 3) of the kinase-like molecule Tribble in Drosophila, and also refers to a gene corresponding to the protein. It has been reported that TRIB3 is induced by endoplasmic reticulum stress as shown in FIG. 3 and increases the activity of TRIB3 promoter in a CHOP and ATF4-dependent manner induced by endoplasmic reticulum stress. Overexpression of TRIB3 increases cell death due to endoplasmic reticulum stress, and conversely, knockdown of TRIB3 reduces cell death. Examination of the functional relationship between CHOP and TRIB3 revealed that TRIB3 is intracellularly bound to CHOP by immunoprecipitation. Overexpression of TRIB3 significantly inhibits the binding of CHOP to the transcriptional conjugating factor p300. These results suggest that TRIB3 functions as an intracellular antagonist to the p300-binding domain of CHOP and suppresses transcription of CHOP. In addition, the stability of ATF4 was reduced by overexpression of TRIB3, and the transcriptional activity was reduced by the same control mechanism as CHOP. In particular, since some genes necessary for survival are known downstream of ATF4, it has been reported that inhibition of ATF4 transcriptional activity due to overexpression of TRIB3 greatly contributes to the induction of apoptosis associated with endoplasmic reticulum stress. (Summary of Research Results Report 2005 by Kakenhi, Nagoya City Hidetoshi Obayashi "Molecular Base of CHOP-Dependent Cell Death in External Stress").

The "CD4 ⁺ CADM1 ⁺ cells" of the present invention are CD4-positive and CDM1- (Cell Addition Molecular 1) -positive T cells that can be detected and separated from HTLV-1-infected PBMC by immunostaining or flow cytometry, and are HTLV. -1 Shows infected T cells. MK-2048 selectively promotes endoplasmic reticulum stress and induces apoptosis in HTLV-1-infected T cells having this cell surface marker. As a result, HTLV-1-infected T cells (CD4 ⁺ CADM1 ⁺ cells) in PBMC infected with HTLV-1 are selectively apoptotic, killed and eliminated by MK-2048. Then, in the PBMC infected with HTLV-1, T cells not infected with HTLV-1 survive. In addition, since MK-2048 does not affect the survival rate of normal PBMC, it has no effect on HTLV-1 non-infected T cells and the like, and cell proliferation of HTLV-1 non-infected T cells and the like proceeds. Therefore, it is effective in preventing the onset of HTLV-1 infected patients and treating the onset patients.

The "apoptosis-promoting agent for HTLV-1-infected PBMC" of the present invention is a drug or a composition containing the drug that promotes apoptosis of HTLV-1-infected PBMC. The apoptosis-promoting agent of the present invention utilizes endoplasmic reticulum stress-mediated apoptosis-inducing action on HTLV-1-infected PBMC, which was found as a novel action of MK-2048. The apoptosis-promoting agent of the present invention selectively induces apoptosis in HTLV-1-infected lymphocytes (T cells positive for CD4 and CADM1) among HTLV-1-infected PBMCs. Thereby, HTLV-1-infected T cells can be selectively killed and eliminated.
The "adult T-cell leukemia (ATL) and / or HTLV-1-related disease prophylactic or therapeutic agent" of the present invention refers to HTLV-1 infection due to the selective apoptosis action of MK-2048 on the above-mentioned HTLV-1-infected PBMC. A pharmaceutical composition that prevents or treats ATL and HTLV-1-related diseases, which are HTLV-1-infected diseases, by killing T cells and eliminating them from peripheral blood. The pharmaceutical composition comprises a pharmaceutically acceptable salt of MK-2048 and / or MK-2048.
These pharmaceutical compositions can be used as oral preparations and parenteral preparations (injections, etc.) depending on the purpose of use, and these preparations are produced by prescribing the above active ingredients in the same manner as ordinary pharmaceutical preparations. can.
Specific examples of such preparations include oral preparations such as tablets, capsules, granules, powders and syrups, injections such as intravenous injection and intramuscular injection, intravenous drip infusions and the like, and these preparations are usually used. It is prepared using the medicinal carrier used.

As the pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the compound of the present invention is used. Specific examples of pharmaceutical carriers used in the production of tablets, capsules, granules and powders include excipients such as lactose, corn starch, sucrose, mannitol, calcium sulfate, crystalline cellulose, carmellose sodium and modified starch. , Disintegrants such as carmellose calcium, binders such as methylcellulose, gelatin, gum arabic, ethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, lubricants such as light anhydrous silicic acid, magnesium stearate, starch, hardened oil Can be mentioned. The tablets may be coated by a well-known method using a usual coating agent.
Specific examples of carriers used in the production of syrups include sweeteners such as sucrose, glucose and fructose, suspending agents such as gum arabic, tragant, sodium carmellose, methyl cellulose, sodium alginate, crystalline cellulose and bee gum. Dispersants such as sorbitan fatty acid ester, sodium lauryl sulfate, polysorbate 80 can be mentioned.
Injectable preparations are usually prepared by dissolving the above active ingredients in distilled water for injection, but if necessary, lysis aids, buffers, pH adjusters, isotonic agents, soothing agents, preservatives, etc. Can be added. Further, the compound may be in the form of a suspended injection in which the compound is suspended in distilled water for injection or vegetable oil, and a base, a suspending agent, a viscous agent and the like can be added as needed.

Hereinafter, the present invention will be described in more detail based on Examples and Test Examples, but the present invention is not limited thereto.

Growth inhibitory effect of HIV integrase inhibitor on HTLV-1-infected T cells, etc. The HIV integrase inhibitor (BMS-707035, RAL, EVG, DTG, CAB, MK-2048) shown in FIG. 1 is an HTLV-1-infected cell line. (KK1, ED, SLB-1, C8166), HTLV-1 non-infected T cell leukemia cell lines (Jurkat and MALT-4), and healthy human PBMC (peripheral blood mononuclear cells) were evaluated for their effects on proliferation.
(1) Material a) HTLV-1-infected T cells, etc .:
Interleukin-2 (IL-2) independent ATL-derived cell lines (OATL4 and ED), HTLV-1-infected cell lines (MT-2, MT-4, SLB-1 and C8166), and HTLV-1 uninfected T Cell leukemia cell lines (Jurkat and MALT-4) were maintained by adding 10% fetal bovine serum to RPMI-1640 medium. The IL-2 dependent ATL cell lines (KOB and KK1) were maintained by adding 100 ng / mL recombinant human IL-2 (manufactured by Peprotec) to the same medium.
b) Healthy human T cells, etc .:
Commercially available products were used for healthy human peripheral blood mononuclear cells (PBMC) (manufactured by Kurabo) and CD4 + T cells (manufactured by Lonza).
c) HTLV-1 infection asymptomatic T cells, etc .:
PBMC samples of 4 HTLV-1 infected asymptomatic patients (mean age 57 years, 33-72 years) were provided by the Institute of Medical Science, University of Tokyo. PBMCs were isolated from blood by density gradient centrifugation using Histpack-1077 (Sigma-Aldrich), followed by RPMI-1640 supplemented with 10% fetal bovine serum and 100 ng / mL human IL-2. Was cultured in.
d) HIV integrase inhibitor:
Examples of HIV integrase inhibitors include BMS-707035 (BMS), raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), cabotegravir (CAB), and MK-2048 in FIG. ) Obtained from. These drugs were dissolved in dimethyl sulfoxide (DMSO) and stored at -80 ° C.
(2) method of each cells were plated approximately 0.75 to 1.5 × 10 ⁵ per well in 24-well plates in the presence of HIV integrase inhibitors (0～100MyuM), or absence 4 days It was cultured. Cell viability analysis was performed using the CellTiter-Glo luminescent cell viability assay and the Glomax detection system (Promega) according to the manufacturer's protocol.
(3) Results As shown in FIG. 2, BMS-707835 and RAL did not significantly inhibit the growth of HTLV-1-infected cells. Although EVG and CAB inhibited the growth of HTLV-1-infected cell lines, they were also cytotoxic to healthy PBMCs. DTG and MK-2048 had a very high growth inhibitory effect on the HTLV-1-infected cell line together with the non-infected leukemia cell line, but the inhibitory effect was not observed in healthy PBMC.
In particular, unlike other HIV integrase inhibitors, MK-2048 showed a stronger growth inhibitory effect on HTLV-1-infected T cell lines as compared with HTLV-1-infected T cell lines. Thus, it was found that MK-2048 has a specific growth inhibitory effect on HTLV-1-infected T cells.

Evaluation of growth inhibitory effect of MK-2048 on HTLV-1-infected T cells, etc. In order to confirm the specific growth inhibitory effect of MK-2048, HTLV-1 non-infected leukemia cells, HTLV-1-infected cells including ATL cells, and healthy patients The growth inhibitory effect of MK-2048 (0 to 50 μM) on human PBMC and CD4 ^{+ T cells was evaluated.}
(1) Material The same cells as in Example 1 were used.
(2) Method The cell viability was measured in the same manner as in Example 1. For the IC50 value (μmol / L) of the growth inhibitory effect, MK-2048 was added to the cell line and cultured for 72 hours, and the viability of the HTLV-1-infected cell line and the non-infected cell line was evaluated.
(3) Results As shown in FIG. 4, MK-2048 strongly suppressed the growth of HTLV-1-infected cell lines (SLB-1, MT-2, MT-4 and C8166) in a dose-dependent manner.
As shown in FIG. 5, MK-2048 was compared with HTLV-1-infected leukemia cell lines (Jurkat and MALT-4) and HTLV-1-infected cell lines (SLB-1, MT-2, MT-4 and). The IC50 value was lower than that of C8166), and it showed a selective growth inhibitory effect on HTLV-1-infected cell lines.
MK-2048 showed a strong growth inhibitory effect on ED and KK1 of the ATL cell line, but did not show a strong growth inhibitory effect on OATL4 and KOB of the ATL cell line. From this, it was found that the growth inhibitory effect of MK-2048 was changed on the ATL cell line (see FIGS. 4 and 5).

Elucidation of the mechanism of action of MK-2048 on MK-2048-sensitive HTLV-1-infected cells In order to elucidate the mechanism of action of MK-2048 in Examples 1 and 2, caspase-3 / 7 activity and cell viability were measured. , The apoptosis-inducing effect of MK-2048 on HTLV-1-infected cells was examined.
Caspases-3, 6 and 7 are proteolytic enzymes classified in the cysteine protease family, and activated caspases include caspases downstream of the signaling pathway, nuclear proteins, membrane proteins, mitochondrial proteins and the like. It degrades many substrate proteins and eventually induces cells to die.
(1) Material The HTLV-1-infected cell line (KK1, ED, SLB-1, OATL4 and KOB) used in Example 1 was used. The MK-2048 sensitive strains are KK1, ED and SLB-1, and the MK-2048 resistant strains are OATL4 and KOB.
(2) approximately 1.5 × 10 ⁵ cells of a method HTLV-1 infected cell lines were seeded into each well of a 24 well plate. HTLV-1-infected cell lines were treated for 24 hours in the presence or absence of 10, 25 or 50 μM of MK-2048. Caspase-3 / 7 activity was measured using the Caspase-Glo 3/7 assay using the Glomax detection system according to the manufacturer's protocol.
(3) Results As shown in FIGS. 6 (A) and 6 (B), MK-2048 activates caspase-3 / 7 and MK-2048-sensitive HTLV-1 infected cell lines (KK1, ED and SLB-1). In cells), cell death was induced in a dose-dependent manner of MK-2048. In contrast, in MK-2048 resistant HTLV-1-infected cell lines (OATL4 and KOB), no activation of caspase-3 / 7 and reduced cell viability by MK-2048 was observed.
From the above, it was clarified that MK-2048 induced cell death in MK-2048-sensitive HTLV-1-infected cells.

Elucidation of the mechanism of action of MK-2048 that induces apoptosis of HTLV-1-infected cells In order to clarify the mechanism of action of MK-2048 that induces apoptosis in HTLV-1-infected cells according to Example 3, MK Treatment was performed for 16 hours with and without the addition of −2048, and how the transcript of the HTLV-1-infected cell line was altered was evaluated by a gene microarray.
(1) Materials MK-2048-sensitive HTLV-1 infected cell lines (KK1, SLB-1 and ED) and MK-2048-resistant HTLV-1 infected cell lines (OATL4) were used according to Example 1.
(2) Method The cells were treated in the presence or absence of 25 μM MK-2048 for 16 hours. Total RNA was isolated using the RNeasy mini kit (manufactured by Qiagen). RNA quality was evaluated by Filmen using an Agent2100 bioanalyzer. Microarray analysis was performed by Filgen using the Afficometrics Human Clarium ™ S Assay. Expression-variable genes were identified by Student's t-test (P <0.01, fold change (FC)> 2.0). The route analysis is performed by the microarray data analysis tool Ver. Using 3.2 (Filgen), genes whose expression fluctuated by 2.0 times or more (P <0.01) were evaluated.
(3) Results As shown in FIG. 7 (A), the gene whose expression was significantly observed in the MK-2048-sensitive HTLV-1-infected cell line (KK1, SLB-1, ED) treated with MK-2048. The numbers were 215, 362 and 33, respectively.
As shown in FIG. 7 (B), among the genes whose expression was significantly observed, 19 were common to MK-2048-sensitive cell lines. In particular, CHAC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) is a novel apoptotic component among genes related to endoplasmic reticulum stress response, and its expression is most significantly increased in MK-2048-sensitive HTLV-1-infected cell lines. Was.

On the other hand, in the MK-2048-resistant HTLV-1-infected cell line (OATL4), MK-2048 did not cause a significant change in gene expression.
Next, a pathway analysis was performed to elucidate the mechanism of action of the growth inhibitory action of MK-2048 on HTLV-1-infected cells. In all MK-2048-sensitive HTLV-1-infected cell lines, treatment with MK-2048 was found to significantly affect the pathway of endoplasmic reticulum stress response, as shown in Table 1 below.

In addition, as shown in Table 2 below, MK-2048 strongly expressed and increased the expression of genes related to endoplasmic reticulum stress response in MK-2048-sensitive HTLV-1-infected cell lines (KK1, SLB-1 and ED).

The endoplasmic reticulum stress response is transmitted primarily through three different signaling pathways: activation of IRE-1α / ERN1, PERK / EIF2AK3 and ATF6, as shown in FIG. In the microarray analysis of MK-2048-sensitive HTLV-1-infected cell lines, as shown in Table 2 above, both CHAC1 and CHOP were markedly upregulated, and these genes were found downstream of the PERK / EIF2AK3 signaling pathway. It is a gene that exists.
Increased expression of the CHAC1 and CHOP / DDIT3 genes occurs when endoplasmic reticulum stress is chronic or severe, and activation of these PERK / EIF2AK3 signaling pathways induces apoptosis.
From the results of the above microarray analysis, it was clarified that MK-2048 induces apoptosis in MK-2048-sensitive HTLV-1-infected cell lines via endoplasmic reticulum stress (via activation of CHAC1 and CHOP). rice field.

Evaluation of Increased Expression of CHOP Gene in HTLV-1-Infected Cells by Treatment with MK-2048 In order to verify the results of microarray analysis in Example 4, MK-2048 was found to be the CHOP gene in HTLV-1-infected cells using quantitative real-time PCR. Confirm that the expression is increased.
(1) Materials MK-2048-sensitive HTLV-1-infected cell lines (KK1, SLB-1, MT-2 and ED) and MK-2048-resistant HTLV-1-infected cell lines (OATL4, KOB) described in Example 1 , HTLV-1 non-infected cell line (Jurkat, MALT-4) was used according to Example 1.
(2) Method Using a part of the total RNA obtained in Example 4, cDNA was constructed using the SuperScriptII First Strand Synthesis System (Thermo Fisher), and the CHOP gene was subjected to quantitative real-time PCR. The expression level was quantified.
Quantitative RT-PCR was performed using a 7500 Fast Real-Time PCR system (Applied Biosystems) according to the manufacturer's protocol. The mRNA level was ^{calculated by the 2-ΔΔCT} method and expressed as a relative ratio compared to the mRNA level of MK-2048 untreated control cells. The primer sequences are listed in Table 3 below.

(3) Results As shown in FIG. 8, MK-2048 is a MK-2048-sensitive HTLV-1-infected cell line (SLB-) as compared with MK-2048-resistant HTLV-1-infected cell lines (OATL-4 and KOB). 1, MT-2, KK1, ED) markedly increased the gene expression of CHOP (P <0.005). The difference was also significant compared to the HTLV-1 non-infected T cell lines (Jurkat and MOLt-4) (P <0.01).
From the above, MK-2048 can increase the gene expression of CHOP via the PERK pathway of endoplasmic reticulum stress in MK-2048-sensitive HTLV-1-infected cell lines, and as a result, induce apoptosis. confirmed.

Confirmation test that apoptosis induced by MK-2048 through endoplasmic reticulum stress (PERK pathway) can be suppressed by PERK inhibitor In Example 5, apoptosis induced by MK-2048 is caused by the PERK pathway of endoplasmic reticulum stress. I showed that. To verify this, it will be examined whether the addition of the PERK signal inhibitor GSK2606414 can suppress the apoptosis induced by MK-2048.
(1) Material An MK-2048-sensitive HTLV-1-infected cell line (ED cell) was used.
The endoplasmic reticulum stress PERK pathway inhibitor GSK2606414 was purchased from Merck & Co., Ltd. and stored at −20 ° C. until dissolved in dimethyl sulfoxide for use.
(2) Method According to Example 3, MK-2048 was added to MK-2048-sensitive HTLV-1-infected cell line ED cells and cultured. At the same time, GSK2606414, which is an inhibitor of the endoplasmic reticulum stress PERK pathway, was added to evaluate changes such as inhibition of ED cell proliferation. The system to which MK-2048 was not added was used as a control.
Changes in cell viability, caspase-3 / 7 activity and mRNA expression levels of CHAC1 and CHOP with and without MK-2048 addition were assessed 24 hours after treatment with 50 μM MK-2048. The values shown in FIG. 9 indicate the cell ratio of the system with or without addition of MK-2048. ED cells were pretreated with GSK2606414 at the indicated concentration 1 hour prior to the addition of 50 μM MK-2048. Control cells were also treated with the indicated concentration of GSK2606414.
(3) Results As shown in FIG. 9, the activation of caspase 3/7 and the decrease in cell viability induced by the treatment with MK-2048 were significantly suppressed by the PERK inhibitor GSK2606414. In addition, inhibition of the PERK pathway with the PERK inhibitor GSK2606414 markedly suppressed the increased expression of the endoplasmic reticulum stress proapoptotic genes CHOP and CHAC1 induced by MK-2048 treatment. From this, it was clarified that the apoptotic cell death induced by MK-2048 can be effectively suppressed by inhibiting the PERK pathway of endoplasmic reticulum stress. On the other hand, in ED cells not treated with MK-2048, caspase 3/7 activation and cell viability were not affected by the addition of the PERK inhibitor GSK2606414.
These results revealed that MK-2048 induces apoptosis of HTLV-1-infected cells through activation of the endoplasmic reticulum stress response (PERK pathway).

Examination of indicators for distinguishing between MK-2048-sensitive strains and resistant strains in HTLV-1-infected cells Regarding GRP78, increased expression was observed in some malignant cancers, leukemias, and multiple myeloma, and multidrug resistance It has been reported to be closely related to poor prognosis (Lee AS. Nat Rev Cancer. 2014; 14 (4): 263-276. Thornton M, Aslam MA, Tweedle EM, et al. Int J Cancer. 2013. 133 (6): 1408-1418. Ways, Luo B, Tseng CC, et al. Blood. 2012; 119 (3): 817-825. Abdel Malek MA, Jagananthan S, Make e, et al. 2015; 6 (5): 3098-3110.)
Therefore, the expression level of GRP78 in HTLV-1-infected cells was measured, and it was evaluated whether the difference between MK-2048-sensitive cells and resistant cells could be discriminated by GRP78.
(1) Materials MK-2048-sensitive strains (SLB-1, MT-2, KK1, ED) and resistant strains (OATL-4 and KOB) of HTLV-1-infected cells, HTLV-1 non-infected T cell lines (Jurkat and MALT-4) was used.
(2) Method According to the method described in Example 5, the mRNA expression level of GRP78 of each cell line was measured, and the mRNA level expressed in Jurkat cells was expressed as a relative ratio of 1.
(3) Results As shown in FIG. 10, in the MK-2048 resistant cell lines (OATL4 and KOB), the mRNA expression level of GRP78 was significantly higher than that in the MK-2048 sensitive strain (P <0.005). ).
This suggests that the sensitivity of MK-2048 is related to the expression level of GRP78. It has been shown that GRP78 may be a discriminant index for the sensitivity and resistance of MK-2048.
In addition, since the expression of GRP78 is decreased in the HTLV-1-infected cell lines (MT-2, ED), it is suggested that the expression of GRP78 is decreased by the HTLV-1 infection.

Comparison of GRP78 mRNA gene expression level between HTLV-1-infected cells and non-infected cells in asymptomatic HTLV-1-infected person PBMC As described in Example 7, it is suggested that the expression of GRP78 is reduced by HTLV-1 infection. Will be done. Therefore, HTLV-1-infected cells and non-infected cells in PBMC obtained from asymptomatic HTLV-1-infected persons were separated, and their GRP78 expression levels were compared.
The HTLV-1 Analysis System (HAS) -Flow method was used to isolate HTLV-1-infected cells, and PBMC of asymptomatic HTLV-1-infected individuals was combined ^{with CD4 + CADM1 +} cell population (HTLV-1-infected cells concentrated). The ^{cells were separated into CD4 +} CADM1 ^- cell populations (enriched with HTLV-1 non-infected cells), and the GRP78 gene expression level in each cell population was measured.
Previous studies have shown that HTLV-1-infected cells are efficiently enriched into ^{the CADM1 + fraction in the CD14-} CD4 ⁺ T cell population. The HAS-Flow method is a method for efficiently concentrating HTLV-1-infected cells by combining these cell surface markers. CADM1 is highly expressed in ATL cells, but not in normal blood cells, and is not highly expressed in other hematological malignancies except for a small part. It is a factor and is considered to be an excellent cell marker for HTLV-1-infected cells.
(1) Material According to the method described in Example 1, PBMC isolated by density gradient centrifugation from blood samples obtained from 9 asymptomatic HTLV-1 infected persons was used.
(2) Method As a cell selection procedure, a combination of biotinylated anti-CADM1, allophycocyanin (APC) -anti-CD7, APC-Cy7-anti-CD3, Pasifwick Blue-anti-CD4 and Pasifwick Orange-anti-CD14 antibody is used for PBMC. Stained using. After staining, the cells were washed and labeled with phycoerythrin-bound streptavidin. Propidium iodide (PI, manufactured by Sigma-Aldrich) was added to the sample and the dead cells were stained immediately prior to flow cytometry. Labeled cells were screened using a FACSAria device (manufactured by BD immunocytometry Systems). Based on CD4 and CADM1 pattern, HTLV-1 infected cell population ^{(PI - / CD14 - / CD3} + / CD4 + / CADM1 +) and non-infected cell populations ^{(PI - / CD14 - / CD3} + / CD4 + / CADM1 -) Fluorescent cells were screened for.
Evaluation of GRP78 gene expression ^levels, CD4 + CADM1 ^- shown as the gene expression ratio of ^CD4 + CADM1 ⁺ cell population for cell populations.
(3) Results As shown in FIG. 11, in 9 samples obtained from asymptomatic HTLV-1-infected persons, the gene expression of GRP78 ^{was enriched by CD4 +} CADM1 ^- fraction (HTLV-1 non-infected cells). ), The CD4 ⁺ CADM1 ⁺ fraction (HTLV-1-infected cells were concentrated) was significantly reduced. Thus, it was found that the gene expression of GRP78 was suppressed in the T cells of HTLV-1-infected patients. In most viral infections other than HTLV-1, the gene expression of GRP78 is gene-amplified and resistant to endoplasmic reticulum stress, but in the case of HTLV-1 viral infection, the opposite is true. Was there. Thus, it was found that the virus infection of HTLV-1 has different characteristics from other virus infections.
In addition, it was suggested that the expression of GRP78 was regulated by HTLV-1 infection, and that infected T cells of asymptomatic HTLV-1 infected individuals were MK-2048 sensitive, and HTLV- was used using MK-2048. It was expected that 1 infected T cells could be specifically eliminated.

Comparison of GRP78 Gene Expression Levels by Disease Type in ATL Patients As shown in Example 8, the expression of GRP78 in asymptomatic HTLV-1-infected T cells was lower than that in non-infected T cells. Therefore, it is expected that the expression of GRP78 is decreased even in ATL patients. In addition, since the prognosis of ATL differs greatly depending on the disease type, it is possible that the expression level of GRP78 differs depending on the disease type. Therefore, the GRP78 gene expression level of ATL patients was compared for each disease type using the gene expression data set accumulated in the gene expression information database.
^{(1) Materials Gene expression of CD4 +} cells of ATL patients (smoldering type: 3 persons, chronic type: 20 persons, acute type: 26 persons) and 21 healthy persons from NCBI Gene Expression Omnibus (GEO), a public gene expression database. Obtained and used a dataset.
(2) Method The expression of GRP78 was summarized in a box whiskers graph for each type ^{of CD4 +} cells and ATL from the data set obtained from the gene expression database.
(3) Results As shown in FIG. 12, the gene expression of GRP78 was significantly reduced in ATL cells as compared with ^{CD4 + cells in healthy subjects.} On the other hand, when viewed by disease type, the expression level of GRP78 was highly malignant, and a large variation was observed in the acute type in which drug resistance was often observed, suggesting control of endoplasmic reticulum stress response by carcinogenesis.
As described above, GRP78, which is a controlling factor of endoplasmic reticulum stress, is significantly reduced in PBMC of ATL patients, and CD4 ⁺ CADM1 ⁺ cell population (HTLV-1-infected cells are concentrated) in PBMC of asymptomatic HTLV-1-infected persons. ) But it decreased significantly. This indicates that HTLV-1-infected T cells are extremely sensitive to endoplasmic reticulum stress-induced apoptosis.
From the data of the present invention, it was revealed that HTLV-1 infection reduces the gene expression of GRP78 in T cells and increases the sensitivity to apoptosis induced by endoplasmic reticulum stress. Therefore, in HTLV-1-infected T cells, the action of MK-2048 enhances the endoplasmic reticulum stress response and induces apoptosis more rapidly.

Evaluation of Efficacy of MK-2048 in Asymptomatic HTLV-1 Infected Persons In order to examine the efficacy of MK-2048 in asymptomatic HTLV-1-infected cells, the indicated concentration for PBMC in asymptomatic HTLV-1-infected persons. Was treated with MK-2048 for 4 days, while untreated PBMC was used as a control (DMSO added). The amount of each HTLV-1 provirus (the amount of viral gene integrated into cell DNA) and the number of cells were measured.
(1) Material PBMC (# 1 to 4) obtained by density gradient centrifugation from the blood of asymptomatic HTLV-1 infected persons (4 persons) was used.
(2) Method Approximately 1 × 10 ⁶ cells of the above PBMC (# 1 to 4) were seeded in each well of a 24-well plate. The seeded PBMCs were treated with or without MK-2048 (25 or 50 μM) for 0 and 4 days, and genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (manufactured by Qiagen). The number of copies of provirus DNA was measured by quantitative real-time PCR using the 7500 Fast Real-Time PCR system. In PCR, the pX region of the HTLV-1 provirus and the human RNase P gene as an internal standard were measured. The amount of provirus was calculated as (2 × pX number of copies) / (PNaseP copy number) and expressed as the number of copies per 100 cells. The number of copies of pX in each sample (# 1-4) was determined in comparison with the number of copies of TL-Om1 cells.
The bar graph of the measurement results shows the average value of the relative ratio of the provirus amount of the MK-2048-treated cells (samples # 1 to 4) to the MK-2048 untreated cells. The number of viable cells was measured at the same time, and the bar graph represents the average value of the relative ratio of the number of cells of MK-2048 treated cells (samples # 1 to 4) to that of MK-2048 untreated cells.
(3) Results As shown in FIG. 13 (A), the amount of provirus was significantly reduced in all PBMCs in a dose-dependent manner when MK-2048 was added. Also, as shown in FIG. 13 (B), MK-2048 does not affect the number of PBMC cells in culture. This indicates that MK-2048 selectively killed HTLV-1-infected cells in PBMC.
These results indicate that MK-2048 selectively eliminates HTLV-1-infected cells in PBMCs and does not affect the survival of normal PBMCs not infected with HTLV-1.

Confirmation that MK-2048 induces endoplasmic reticulum stress-induced apoptosis specific to HTLV-1-infected cells In Example 10, MK-2048 selectively eliminated HTLV-1-infected cells in PBMC and HTLV- It was shown that it does not affect the survival of normal PBMCs not infected with 1. Therefore, in this example, it was confirmed whether or not MK-2048 induces endoplasmic reticulum stress-induced apoptosis in a HTLV-1-infected cell-specific manner.
(1) Material PBMC obtained by density gradient centrifugation from the blood of an asymptomatic HTLV-1 infected person was used.
(2) Method Approximately 1.5 × 10 ⁵ cells were seeded on a 24-well plate and treated with MK-2048 (25 μM) for 24 hours.
For staining of CHOP, a marker molecule for induction of endoplasmic reticulum stress apoptosis, cells were attached to MAS-coated glass slides, methanol-fixed at −20 ° C. for 15 minutes, blocked with antibody blocks (Agilent Technologies), and blocked. After incubation with the primary antibody, it was detected with the secondary antibody. The cover glass was then attached using an encapsulant containing DAPI and photographed with an Olympus FV1000 confocal microscope. The antibodies used are listed in Table 4 below.

For apoptotic cell analysis, PBMCs were stained with a mixture of biotin anti-CADM1 antibody, FITC anti-CD14 antibody, and PE anti-CD4 antibody. Further, the cells were stained with streptavidin APKCy7 antibody, APC anti-Annexin V antibody, and DAPI, and the stained PBMCs were analyzed using CytoFLEX (Beckman Coulter). FlowJo software (TreeStar) was used for the analysis.
(3) Results As shown in FIG. 14 (A), ^{expression of CHOP was observed in CD4 +} CADM1 ⁺ cells (HTLV-1-infected cells), but CD4 ⁺ CADM1 ^- cells (HTLV-1-non-infected cells). No ^{expression of CHOP was observed in CD4-} CADM1 ^- cells (cells other than T cells).
Figure 14 is the (B) ^CD4 + CADM1 ^- indication of fractions compared (HTLV-1 uninfected cells concentration) ^and, CD4 + CADM1 ⁺ fraction (HTLV-1 infected cells enriched) at significantly induce apoptosis It has been shown that the amount of Annexin V is increased. Figure 14 shows the (C) in 6 specimen extracting analysis results by boxplot ^graph, CD4 + CADM1 ^- compared to fraction (HTLV-1 uninfected cells ^enrichment), CD4 ⁺ CADM1 + fraction (HTLV It has been shown that the amount of annexin V is significantly increased in -1 infected cells (enriched) (P <0.005).

Considering the results of the present invention together, MK-2048 induces a endoplasmic reticulum stress response in PBMC of asymptomatic patients with HTLV-1 infection, induces apoptosis involved in the PERK pathway, and infects HTLV-1. It was revealed that T cells were selectively killed and eliminated.
Thus, since MK-2048 selectively induces PERK-related apoptosis of endoplasmic reticulum stress in HTLV-1-infected T cells, MK-2048 can be used as a therapeutic agent for ATL and HTLV-1-related diseases. ..
In addition, MK-2048 can be used as a drug for endoplasmic reticulum stress targeting therapy due to its mechanism of action, and because of its novel mechanism of action, it is used in combination with other anti-HTLV-1 agents for ATL and HTLV-1-related diseases. And the synergistic effect can be expected.
Furthermore, MK-2048 has a high safety profile, indicating that it is useful for elderly adult patients with ATL.

The novel action of MK-2048 found in the present invention induces endoplasmic reticulum stress response in HTLV-1-infected T cells, induces apoptosis involved in the PERK pathway, and selectively selects HTLV-1-infected T cells. It kills and eliminates. Therefore, MK-2048 can be used as a therapeutic agent for a new mechanism of action for ATL and HTLV-1-related diseases. Since the endoplasmic reticulum stress targeting therapy with the apoptosis involved in the endoplasmic reticulum stress as the mechanism of action is novel, it is used in combination with an anti-HTLV-1 agent having another mechanism of action in ATL and HTLV-1-related diseases. A synergistic effect can be expected. In addition, MK-2048 has a high safety profile and is therefore useful for elderly adult patients with ATL.

Claims (16)

An endoplasmic reticulum stress promoter for HTLV-1-infected peripheral blood mononuclear cells (PBMC) containing MK-2048 as an active ingredient. The endoplasmic reticulum stress promoter according to claim 1, wherein the PBMC is a T cell. The endoplasmic reticulum stress promoter according to claim 1 or 2, wherein the HTLV-1-infected PBMC is CD4 ⁺ CADM1 ^{+ cells.} The endoplasmic reticulum stress promoter according to any one of claims 1 to 3, wherein the endoplasmic reticulum stress promotion is promotion of gene expression in the PERK pathway. The endoplasmic reticulum stress promoter according to claim 4, wherein the gene is CHAC1, TRIB3 or CHOP / DDIT3. The endoplasmic reticulum stress promoter according to claim 4 or 5, wherein the gene is CHOP / DDIT3. An apoptosis-promoting agent for HTLV-1-infected PBMC containing MK-2048 as an active ingredient. The apoptosis-promoting agent according to claim 7, wherein the PBMC is a T cell. The apoptosis-promoting agent according to claim 7 or 8, wherein the HTLV-1-infected PBMC is CD4 ⁺ CADM1 ^{+ cells.} The apoptosis-promoting agent according to claim 9, wherein the promotion of apoptosis is a decrease in gene expression of GRP78 and an increase in gene expression of CHAC1, TRIB3 or CHOP / DDIT3. The apoptosis-promoting agent according to claim 10, wherein the enhancement of the PERK pathway is an enhancement of CHOP / DDIT3 gene expression. A selective eliminator of HTLV-1-infected PBMC containing MK-2048 as an active ingredient. The selective exclusion agent according to claim 12, wherein the PBMC is a T cell. The selective exclusion agent according to claim 12 or 13, wherein the HTLV-1-infected PBMC is CD4 ⁺ CADM1 ^{+ cells.} A prophylactic or therapeutic agent for adult T-cell leukemia (ATL) and / or HTLV-1-related diseases containing MK-2048 as an active ingredient. The prophylactic or therapeutic agent according to claim 15, wherein the prophylaxis or treatment is the selective elimination of HTLV-1-infected PBMCs.

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