Pharmaceutical composition for treating lymphoma

Abstract

Provided is a method for treating lymphoma. The present invention uses a pharmaceutical composition that is for treating lymphoma and comprises BCV, a pharmaceutically acceptable salt thereof, or a solvate of these. The lymphoma may be EBV-positive lymphoma. The lymphoma may be MYC-positive lymphoma. The pharmaceutical composition may be used in combination with a chemotherapeutic agent.

Description

Pharmaceutical composition for treating lymphoma

[0001] The technical field of the invention relates to the treatment of lymphoma.

[0002] Lymphoma is a type of blood cancer known to occur when lymphocytes become cancerous. Non-Patent Document 1 describes that non-Hodgkin's lymphoma is the most common hematological malignancy in the world, accounting for approximately 3% of cancer diagnoses and deaths. Non-Patent Document 2 describes that the incidence of Hodgkin's lymphoma has been increasing over the past decade, particularly among women, younger people, and people from Asian countries.

[0003] Several reports have been published in recent years regarding methods for treating lymphoma. Non-Patent Document 3 describes the possibility of treatment targeting CD19. Non-Patent Document 4 describes the possibility of treatment targeting EZH2.

[0004] "Epidemiology of Non-Hodgkin's Lymphoma" Thandra et al., Med Sci (Basel). 2021 Jan 30;9(1):5."Incidence, mortality, risk factors, and trends for Hodgkin lymphoma: a global data analysis" Huang et al., J Hematol Oncol. 2022 May 11;15(1):57."Targeting CD19 for diffuse large B cell lymphoma in the era of CARs: Other modes of transportation" Blood Rev. 2022 Aug 17;101002."Taking the EZ way: Targeting enhancer of zeste homolog 2 in B-cell lymphomas" Morschhauser et al., Blood Rev. 2022 Jul 9;100988.

[0005] Although various findings have been accumulated to date, many aspects of the mechanisms of lymphoma remain unknown, and conventional treatment approaches alone have not been sufficient.

[0006] Meanwhile, the present inventors have conducted extensive research and discovered that brincidofovir (hereinafter referred to as BCV) exhibits excellent effects in inhibiting the proliferation of lymphoma cells.

[0007] That is, one aspect of the present invention provides a pharmaceutical composition for treating lymphoma, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. This pharmaceutical composition can be used to treat lymphoma.

[0008] Figures 1A-C show the results of investigating the effect of BCV treatment on lymphoma cell proliferation. Figures 1A and 1B show the results using EBV-positive cells, and Figure 1C shows the results using EBV-negative cells. Figure 2 shows the results of investigating the effect of BCV or various antiviral drug treatment on the proliferation of EBV-positive NK / T lymphoma cells. Figures 3A-D show the results of administering BCV to NSG mice transplanted with lymphoma cells. Figure 3A shows the results of examining tumor size on days 1, 5, 8, 12, and 15. Figure 3B shows the results of examining body weight on days 1, 5, 8, 12, and 15. Figure 3C shows the results of measuring tumor weight. The values ​​in the bar graphs indicate vehicle administration and BCV administration, from left to right. Figure 3D shows photographs of tumors collected from each individual. Figure 4 shows the results of investigating the effect of BCV treatment on EBNA1 and LMP1 gene expression in lymphoma cells. Figure 5A shows the effect of BCV treatment on Myc gene expression in lymphoma cells. The bar graph values ​​in Figures 5A-C indicate, from left to right, DMSO, 0.1 μg / mL (0.17 μM) BCV, and 1 μg / mL (1.7 μM) BCV. Figures 5B and 5C show the effect of BCV treatment on the expression of STING pathway genes in lymphoma cells. Figure 6 shows the effect of BCV treatment on immunogenic cell death. The bar graph values ​​indicate, from left to right, DMSO, 0.1 μg / mL (0.17 μM) BCV, and 1 μg / mL (1.7 μM) BCV. Figures 7A-C show the effect of BCV and various anticancer drug treatments, or BCV treatment and radiation, on lymphoma cell proliferation. Figure 7A shows the results of etoposide or gemcitabine treatment. Figure 7B shows the results of treatment with BCV, BCV and etoposide, or BCV and gemcitabine. Figure 7C shows the results of treatment with BCV and radiation. Figure 8 shows the results of examining the effect of BCV treatment on the growth of MYC-amplified lymphoma (EBV-positive or -negative). Figure 9 shows the results of examining the sensitivity of 11 lymphoma cell lines to the effects of BCV treatment on growth. Figures 10A and 10B show the results of gene expression analysis. Figure 10A shows the gene set that showed increased expression in BCV-sensitive cell lines. Figure 10B shows an enrichment plot of MYC TARGETS V2. Figures 11A and 11B show the results of examining the prognosis of a human patient cohort.Figure 11A shows the results of investigating progression-free survival, Figure 11B shows the results of investigating overall survival, and Figure 12 shows the results of investigating genes that showed increased expression in the poor prognosis group of a human patient cohort.

[0009] Hereinafter, embodiments of the present invention will be described in detail, with the same contents omitted as appropriate to avoid repetition.

[0010] (1) Treatment Method According to one embodiment of the present invention, there is provided a method for treating lymphoma, comprising administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject. This treatment method enables the treatment of lymphoma through a novel therapeutic approach.

[0011] The lymphoma may be a MYC-positive lymphoma. In this case, BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can provide a particularly excellent therapeutic effect. For example, in the case of MYC-positive lymphoma, a high growth inhibitory effect can be obtained not only against EBV-positive lymphoma but also against EBV-negative lymphoma.

[0012] In the above-mentioned therapeutic method, the subject may be, for example, a MYC-positive subject. In this case, particularly excellent therapeutic effects can be obtained by BCV, its pharmaceutically acceptable salt, or a solvate thereof. For example, in MYC-positive subjects, a high growth inhibitory effect can be obtained not only for EBV-positive lymphoma but also for subjects suffering from EBV-negative lymphoma.

[0013] The above-mentioned treatment method may include, for example, a step of identifying a MYC-positive subject. In this case, particularly excellent therapeutic effects of BCV, its pharmaceutically acceptable salt, or a solvate thereof can be obtained. For example, in MYC-positive subjects, a high growth-inhibitory effect can be obtained not only in subjects with EBV-positive lymphoma but also in subjects with EBV-negative lymphoma.

[0014] In the above-mentioned treatment method, the subject may be a subject identified using, for example, the expression level of MYC as an index. In this case, particularly excellent therapeutic effects can be obtained by BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. For example, identifying and treating a subject with a high expression level of MYC can achieve a greater inhibitory effect on lymphoma proliferation than treating a subject with a low expression level of MYC.

[0015] The above-described treatment method may include, for example, a step of identifying a subject with high MYC expression as a treatment subject. In this case, a particularly excellent therapeutic effect can be obtained by BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. For example, treating a subject with high MYC expression can achieve a greater inhibitory effect on lymphoma proliferation than treating a subject without high MYC expression.

[0016] The above-mentioned treatment method may include, for example, a step of detecting the expression level of MYC in a subject's specimen. In this case, particularly excellent therapeutic effects can be obtained by BCV, its pharmaceutically acceptable salt, or a solvate thereof. For example, a high therapeutic effect can be obtained by treating a subject provided with a specimen having a high MYC expression level. For example, if the subject's specimen has a high MYC expression level, the subject may be identified as a subject to be administered BCV or a low dose of BCV. On the other hand, for example, if the subject's specimen has a low MYC expression level, the subject may be identified as a subject not to be administered BCV or a subject to be administered a high dose of BCV.

[0017] The above-mentioned therapeutic method may include, for example, administering a higher dose of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject provided with a sample having a high MYC expression level than to a subject provided with a sample having a low MYC expression level, thereby achieving a particularly excellent therapeutic effect of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0018] The above-mentioned therapeutic method may, for example, comprise a step of administering brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject, wherein the subject has been shown to have a higher MYC expression level in a specimen from the subject as compared to a specimen previously confirmed not to have high MYC expression, by detecting the expression level of MYC in the specimen from the subject. In this case, particularly excellent therapeutic effects of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can be obtained.

[0019] The above-mentioned treatment method may include, for example, administering brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject whose MYC expression level has been shown to be higher than that of a MYC-negative sample by detecting the expression level of MYC in the subject's sample, thereby achieving a particularly excellent therapeutic effect of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0020] The lymphoma may be EBV-positive lymphoma. In this case, BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof provides a particularly excellent therapeutic effect. For example, a higher growth inhibitory effect is obtained in this EBV-positive lymphoma than in EBV-negative lymphoma.

[0021] In the above-mentioned therapeutic method, the subject may be, for example, an EBV-positive subject. In this case, a particularly excellent therapeutic effect can be obtained by BCV, its pharmaceutically acceptable salt, or a solvate thereof. For example, a higher growth inhibitory effect can be obtained in an EBV-positive subject than in a subject suffering from EBV-negative lymphoma.

[0022] The above-mentioned treatment method may include, for example, a step of identifying an EBV-positive subject. In this case, a particularly excellent therapeutic effect of BCV, its pharmaceutically acceptable salt, or a solvate thereof can be obtained. For example, a higher growth inhibitory effect can be obtained in EBV-positive subjects than in subjects suffering from EBV-negative lymphoma.

[0023] In the above-mentioned treatment method, the subject may be a subject identified using, for example, the expression level of EBV as an index. In this case, particularly excellent therapeutic effects can be obtained by BCV, its pharmaceutically acceptable salt, or a solvate thereof. For example, identifying and treating a subject with a high expression level of EBV can achieve a greater inhibitory effect on lymphoma proliferation than treating a subject with a low expression level of EBV.

[0024] The above-mentioned treatment method may include, for example, a step of identifying a subject with high EBV expression as a treatment subject. In this case, a particularly excellent therapeutic effect can be obtained by BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. For example, treating a subject with high EBV expression can achieve a greater inhibitory effect on lymphoma proliferation than treating a subject without high EBV expression.

[0025] The above-mentioned treatment method may include, for example, a step of detecting the expression level of EBV in a subject's specimen. In this case, particularly excellent therapeutic effects can be obtained by BCV, its pharmaceutically acceptable salt, or a solvate thereof. For example, a high therapeutic effect can be obtained by treating a subject provided with a specimen having a high EBV expression level. For example, if the EBV expression level in the subject's specimen is high, the subject may be identified as a subject to be administered BCV or a subject to be administered a low dose of BCV. On the other hand, for example, if the EBV expression level in the subject's specimen is low, the subject may be identified as a subject not to be administered BCV or a subject to be administered a high dose of BCV.

[0026] The above-mentioned therapeutic method may include, for example, administering a higher dose of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject providing a specimen with a high EBV expression level than to a subject providing a specimen with a low EBV expression level, in which case a particularly excellent therapeutic effect of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can be obtained.

[0027] The above-mentioned therapeutic method may, for example, comprise a step of administering brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject, wherein the subject has been shown by detecting the EBV expression level in a specimen from the subject to be higher than the EBV expression level in a specimen previously confirmed not to have high EBV expression, in which case particularly excellent therapeutic effects of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can be obtained.

[0028] The above-mentioned therapeutic method may include, for example, a step of administering brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject whose EBV expression level has been shown to be higher than that of an EBV-negative sample by detecting the EBV expression level in the subject's sample, in which case a particularly excellent therapeutic effect of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can be obtained.

[0029] The above-mentioned treatment method may include, for example, a step of identifying a MYC-positive and EBV-positive subject as a treatment target, a step of identifying a subject using the expression levels of MYC and EBV as an index, or a step of identifying a subject with high expression of MYC and EBV as a treatment target. In this case, particularly excellent therapeutic effects can be obtained by BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. The above-mentioned lymphoma may be MYC-positive and EBV-positive lymphoma. In this case, particularly excellent therapeutic effects can be obtained by BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. The above-mentioned treatment method may include, for example, a step of identifying a MYC-positive and EBV-negative subject. The above-mentioned lymphoma may be MYC-positive and EBV-negative lymphoma.

[0030] The lymphoma may be, for example, EBV-positive NK / T lymphoma, MYC-positive EBV-positive NK / T lymphoma, MYC-positive Burkitt lymphoma, MYC-positive DLBCL, MYC-positive double-hit DLBCL, or MYC-positive triple-hit DLBCL, in which case BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can provide a particularly excellent therapeutic effect.

[0031] The above-described treatment method may include a step of administering a chemotherapeutic agent to the subject. In this case, the combination provides a particularly excellent therapeutic effect. For example, this combination treatment provides a synergistic effect of BCV and the chemotherapeutic agent. The synergistic effect includes, for example, an effect greater than the sum of the lymphoma growth inhibitory effect obtained by BCV monotherapy and the lymphoma growth inhibitory effect obtained by chemotherapeutic agent monotherapy. The use of an antimetabolite or topoisomerase inhibitor in combination with BCV provides a particularly excellent therapeutic effect. Furthermore, the use of an immune checkpoint inhibitor in combination with BCV provides a particularly excellent therapeutic effect. The chemotherapeutic agent may be administered to the subject before, simultaneously with, or after the administration of BCV. "Simultaneous with administration" includes "simultaneous" and refers to the same period, and may be substantially simultaneous in consideration of standard treatment procedures. "Simultaneous with administration" includes the administration of BCV and the chemotherapeutic agent as a combination drug.

[0032] The above-mentioned treatment method may include a step of irradiating the subject. The irradiation may include, for example, gamma irradiation. The irradiation may be administered to the subject before, simultaneously with, or after the administration of BCV.

[0033] The above-mentioned therapeutic method may involve, for example, administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject to reduce MYC expression in lymphoma cells of the subject. This therapeutic method may improve the prognosis of human lymphoma patients due to reduced MYC expression.

[0034] The above-mentioned therapeutic method may induce immunogenic cell death, for example, by administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject.

[0035] In the above-described therapeutic methods, the subject (including the patient) may be a human or a non-human mammal (e.g., one or more species, such as a mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee). The patient may also be a patient diagnosed with lymphoma (e.g., MYC-positive or EBV-positive lymphoma) or a patient in need of lymphoma treatment. The patient may also be, for example, a patient who has been administered a chemotherapeutic agent, a patient receiving treatment with a chemotherapeutic agent, a patient who has been administered BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a patient receiving treatment with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. Treatment with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof is preferably performed on patients with NK / T-cell lymphoma. In this case, excellent therapeutic effects on NK / T-cell lymphoma, minimizing side effects, reducing MYC expression, and improving prognosis are particularly obtained. In this case, the NK / T cell lymphoma is preferably EBV-positive or highly expresses EBV. In this case, a particularly excellent therapeutic effect on the NK / T cell lymphoma can be obtained. Furthermore, the NK / T cell lymphoma is preferably MYC-positive or highly expresses MYC. In this case, a particularly excellent therapeutic effect on the NK / T cell lymphoma can be obtained.

[0036] The above-mentioned treatment method may include, for example, (i) identifying a MYC-positive or EBV-positive subject as a subject for lymphoma treatment, (ii) identifying a subject with MYC-positive lymphoma or EBV-positive lymphoma as a subject for lymphoma treatment, (iii) detecting MYC or EBV in the subject, (iv) detecting the presence or absence of MYC or EBV in the subject, (v) collecting a subject's sample (e.g., lymphoma cells, lymph node tissue, or a blood sample (e.g., plasma, serum, or whole blood)), (vi) detecting MYC or EBV in the collected sample, or (vii) identifying a subject who provided a sample in which MYC or EBV was detected as a subject for treatment. Using one or more of these steps is useful for administering treatment to a population that will likely respond to treatment. When two or more steps are used, the order of these steps is arbitrary and can be determined depending on the desired treatment method. These steps may be performed before or after the step of administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a chemotherapeutic agent to the subject. Detection may be performed using gene dosage, mRNA dosage, protein dosage, or gene translocation as an indicator. Detection may also be performed by testing.

[0037] The above-mentioned treatment method may include, for example, a step of identifying a subject having lymphoma as a subject for lymphoma treatment, a step of identifying a subject who is positive for lymphoma markers as a subject for lymphoma treatment, a step of administering a therapeutically effective amount of BCV or a chemotherapeutic agent to the subject, a step of suppressing proliferation of lymphoma cells in the subject, a step of reducing lymphoma markers in the subject, a step of reducing expression of MYC in the lymphoma of the subject, or a step of suppressing lymph node swelling in the subject. Additionally, the above-mentioned treatment methods or subjects may optionally include, for example, identifying a subject with a cytomegalovirus (CMV), adenovirus (AdV), BK virus (BKV), or variola virus (VaV) infection, identifying a CMV, AdV, BKV, or VaV-seropositive subject, identifying a subject in need of prevention or treatment of CMV, AdV, BKV, or VaV infection, identifying a subject after allogeneic transplant or allogeneic transplant (e.g., hematopoietic cell transplant), identifying a subject in need of an immunosuppressant, identifying an immunosuppressed subject, a method for treating virus-induced tumors in an immunosuppressed subject, or a method for preventing or treating viral (e.g., CMV, AdV, BKV, or VaV) infection.

[0038] (2) Pharmaceutical Composition According to one embodiment of the present invention, there is provided a pharmaceutical composition for treating lymphoma, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. This pharmaceutical composition enables the treatment of lymphoma through a novel therapeutic approach.

[0039] The lymphoma may be a MYC-positive lymphoma. In this case, BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can provide a particularly excellent therapeutic effect. For example, in the case of MYC-positive lymphoma, a high growth inhibitory effect can be obtained not only against EBV-positive lymphoma but also against EBV-negative lymphoma.

[0040] The lymphoma may be EBV-positive lymphoma. In this case, BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can provide a particularly excellent therapeutic effect. For example, a higher growth inhibitory effect can be obtained in EBV-positive lymphoma than in EBV-negative lymphoma.

[0041] In the above-mentioned treatment, the subject may be a subject identified using, for example, the expression level of EBV or MYC as an index. In this case, particularly excellent therapeutic effects can be obtained by BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. For example, identifying and treating a subject with a high expression level of EBV or MYC can achieve a greater inhibitory effect on lymphoma proliferation than treating a subject with a low expression level of EBV or MYC.

[0042] The above-mentioned treatment may include, for example, detecting the expression level of EBV or MYC in a subject's specimen. In this case, a particularly excellent therapeutic effect can be obtained by using BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. For example, a high therapeutic effect can be obtained by treating a subject who has provided a specimen with a high expression level of EBV or MYC.

[0043] The above-mentioned treatment may include, for example, administering brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject, and the subject may be a subject whose EBV or MYC expression level has been shown to be higher than that of a sample previously confirmed not to have high EBV or MYC expression, as determined by detecting the expression level of EBV or MYC in the sample. In this case, a particularly excellent therapeutic effect can be achieved by BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0044] The lymphoma may be MYC-positive and EBV-positive lymphoma, in which case BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can provide a particularly excellent therapeutic effect.

[0045] The lymphoma may be, for example, EBV-positive NK / T lymphoma, MYC-positive EBV-positive NK / T lymphoma, MYC-positive Burkitt lymphoma, MYC-positive DLBCL, MYC-positive double-hit DLBCL, or MYC-positive triple-hit DLBCL, in which case BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof can provide a particularly excellent therapeutic effect.

[0046] The pharmaceutical composition may be used in a combination therapy of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, and a chemotherapeutic agent. In this case, a particularly excellent therapeutic effect can be obtained by the combination. For example, this combination therapy can provide a synergistic effect by BCV and the chemotherapeutic agent. The synergistic effect includes, for example, an effect exceeding the sum of the lymphoma growth inhibitory effect obtained by treatment with BCV alone and the lymphoma growth inhibitory effect obtained by treatment with the chemotherapeutic agent alone.

[0047] The pharmaceutical compositions include those containing BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof for use in treating lymphoma. From another aspect, there is provided use of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof for the manufacture of a pharmaceutical composition for treating lymphoma.

[0048] According to one embodiment of the present invention, there is provided a pharmaceutical composition for treating lymphoma, comprising BCV, a pharmaceutically acceptable salt or solvate thereof, or a chemotherapeutic agent, for use in combination therapy with BCV, a pharmaceutically acceptable salt or solvate thereof, or a chemotherapeutic agent. As described above, this pharmaceutical composition provides a particularly excellent therapeutic effect when used in combination.

[0049] The pharmaceutical composition includes a pharmaceutical composition for use in the treatment method of (1) above. This treatment method may include at least one of any of the steps of (1) above (e.g., steps (i) to (vii)).

[0050] (3) Inhibition Method According to one embodiment of the present invention, there is provided a method for inhibiting MYC, comprising the step of administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject. The proliferation of MYC-positive lymphoma cells is suppressed by inhibiting MYC. Therefore, this inhibition method can more effectively suppress the proliferation of MYC-positive lymphoma cells. This method may include at least one of the steps in (1) above (e.g., steps (i) to (vii)). This method includes a method for treating a disease by inhibiting MYC. The disease includes, for example, a disease caused by MYC. The disease includes, for example, lymphoma (e.g., MYC-positive lymphoma).

[0051] According to one embodiment of the present invention, there is provided a composition for inhibiting MYC, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. The proliferation of MYC-positive lymphoma cells is suppressed by inhibiting MYC. Therefore, use of this composition can more effectively inhibit the proliferation of MYC-positive lymphoma cells. This composition includes a composition for use in the above-mentioned inhibition method.

[0052] The above-mentioned compositions include compositions containing BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof for use in inhibiting MYC. From another aspect, there is provided use of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof for the manufacture of a composition for inhibiting MYC.

[0053] According to one embodiment of the present invention, there is provided a method for inhibiting MYC, comprising the step of contacting MYC with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, including, for example, in vitro, ex vivo, or in vivo inhibition.

[0054] According to one embodiment of the present invention, there is provided a method for inhibiting EBNA1 or LMP1, comprising the step of administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to a subject. This inhibition method can more effectively suppress the proliferation of EBV-positive lymphoma cells. This method may comprise at least one of the steps (1) above (e.g., steps (i) to (vii)). This method includes a method for treating a disease by inhibiting EBNA1 or LMP1. The disease includes, for example, a disease caused by EBV. The disease includes, for example, lymphoma (e.g., EBV-positive lymphoma). From another perspective, there is provided a composition for inhibiting EBNA1 or LMP1, comprising BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. From another perspective, there is provided use of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof for the manufacture of a composition for inhibiting EBNA1 or LMP1. In another aspect, there is provided a method for inhibiting EBNA1 or LMP1, comprising the step of contacting EBNA1 or LMP1 with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof. The inhibition can be, for example, in vitro, ex vivo, or in vivo.

[0055] (4) Diagnostic Method According to one embodiment of the present invention, a companion diagnostic method is provided, which includes a step of detecting the presence or absence of MYC positivity in a subject. This method includes a diagnostic method for evaluating the effectiveness of treatment with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof prior to administration. This diagnostic method allows treatment to be administered to a population that is likely to respond to treatment. Here, the subject includes a patient suffering from lymphoma or lymphoma in such a patient.

[0056] According to one embodiment of the present invention, there is provided a companion diagnostic method comprising a step of detecting the presence or absence of EBV positivity in a subject. This method includes a diagnostic method for evaluating the effectiveness of treatment with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof prior to administration. This diagnostic method allows treatment to be administered to a population that is likely to benefit from treatment. Here, the subject includes a patient suffering from lymphoma or lymphoma in such a patient.

[0057] According to one embodiment of the present invention, there is provided a companion diagnostic method comprising a step of detecting the presence or absence of MYC-positive and EBV-positive in a subject. This method includes a diagnostic method for evaluating the effectiveness of treatment with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof before administration. This diagnostic method allows treatment to be administered to a population that is likely to respond to treatment. In the diagnostic method, the subject includes a patient suffering from lymphoma or lymphoma in such a patient.

[0058] According to one embodiment of the present invention, there is provided a companion diagnostic method comprising a step of detecting EBV or MYC expression in a subject. This method includes a diagnostic method for evaluating the effectiveness of treatment with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof prior to administration. This diagnostic method allows treatment to be administered to a population that is likely to benefit from treatment. Here, the subject includes a patient suffering from lymphoma or lymphoma in such a patient.

[0059] According to one embodiment of the present invention, a method for diagnosing whether a subject has lymphoma highly susceptible to BCV, its pharmaceutically acceptable salt, or a solvate thereof is provided, comprising a step of examining the expression level of EBV or MYC in a sample from the subject. This method can be used to diagnose whether a subject has lymphoma highly susceptible to BCV using the expression level of EBV or MYC as an indicator. Therefore, use of this method allows for more appropriate BCV treatment. For example, if a subject is diagnosed as having a highly susceptible lymphoma, treatment may be performed by administering BCV, or by administering it at a low dose. On the other hand, for example, if a subject is diagnosed as not having a highly susceptible lymphoma, treatment may be performed by not administering BCV, or by administering it at a high dose. This diagnosis may also be a companion diagnostic for treatment using BCV, its pharmaceutically acceptable salt, or a solvate thereof.

[0060] From another perspective, one embodiment of the present invention provides a kit or composition for use in any of the above diagnostic methods or companion diagnostic methods. This kit or composition includes a means for measuring, for example, the gene dosage, mRNA level, protein level, or gene translocation of MYC or EBV. The measuring means may include, for example, a primer (e.g., a primer capable of amplifying at least a portion of the MYC or EBV gene (e.g., a primer capable of binding to the MYC or EBV gene, or an upstream or downstream site thereof)) or a probe (e.g., a probe capable of detecting a translocation of the MYC gene (e.g., a FISH probe)).

[0061] (5) Other Methods According to one embodiment of the present invention, there is provided a method for identifying subjects for treatment with BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, comprising the step of detecting MYC or EBV in a subject. This method allows treatment to be administered to a population for which treatment is likely to be effective. For example, if a subject has a high expression level of EBV or MYC and is predicted to be highly sensitive, the subject may be identified as a subject for administration of BCV or a low dose of BCV. On the other hand, if a subject has a low expression level of EBV or MYC and is predicted to be less sensitive, the subject may be identified as a subject for which BCV should not be administered or a high dose of BCV. Here, the subject includes a patient suffering from lymphoma or a specimen from such a patient.

[0062] According to one embodiment of the present invention, there is provided a method for improving the prognosis of a subject in the treatment of lymphoma, comprising the step of administering BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof to the subject. BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof has the effect of reducing MYC expression, and therefore can improve the prognosis of a subject in the treatment of lymphoma. An improved prognosis includes, for example, a better prognosis. An improved prognosis includes, for example, an extension of progression-free survival or survival time. An improved prognosis includes, for example, a state in which the prognosis is improved compared to that of a patient administered a treatment method that does not reduce MYC expression. Prognosis includes, for example, a change in a patient's symptoms over the course of the disease.

[0063] According to one embodiment of the present invention, there is provided a method for predicting or assessing the prognosis of a malignant tumor, comprising a step of using the expression level of MYC in a lymphoma subject as an index. This method can be used to predict or assess the prognosis of a patient. For example, a high level of MYC expression in a lymphoma subject's sample may be predicted or assessed as a poor prognosis. For example, a low level of MYC expression in a lymphoma subject's sample may be predicted or assessed as a good prognosis.

[0064] The above embodiments (1) to (5) are described in more detail below. The following detailed embodiments and explanations of each term can be applied to any of the above embodiments (1) to (5).

[0065] In the above (1) to (5), BCV (brincidofovir) includes a compound having the structure represented by the following formula: BCV can also be represented by the IUPAC name of [(2S)-1-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxypropan-2-yl]oxymethyl-(3-hexadecoxypropoxy)phosphinic acid or ({[(2S)-1-(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)-3-hydroxypropan-2-yl]oxy}methyl)[3-(hexadecyloxy)propoxy]phosphinic acid. BCV includes a compound represented by the CAS registry number 444805-28-1. In this specification, BCV is an abbreviation for brincidofovir, and the two terms have the same meaning.

[0066] In the above (1) to (5), lymphoma includes malignant lymphoma. Malignant lymphoma includes, for example, diseases caused by canceration of lymphocytes. Malignant lymphoma includes, for example, non-Hodgkin's lymphoma or Hodgkin's lymphoma. Non-Hodgkin's lymphoma includes, for example, B-cell lymphoma or NK / T-cell lymphoma. B-cell lymphoma includes, for example, follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, primary effusion lymphoma, or chronic lymphocytic leukemia / small lymphocytic lymphoma. Examples of NK / T-cell lymphomas include peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma (nasal type), and cutaneous lymphoma (e.g., mycosis fungoides). Examples of Hodgkin lymphomas include classical Hodgkin lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma. For details on lymphoma, see, for example, Nirmal, J Oral Maxillofac Pathol. 2020 May-Aug;24(2):195-199; Singh et al., J Family Med Prim Care. 2020 Apr;9(4):1834-1840; or Voltin et al., Cancers (Basel). 2020 Mar 5;12(3):601. The therapeutic effect or onset of lymphoma may be diagnosed by examining lymph node swelling using CT, PET, or MRI, histological examination of lymph nodes or tumor masses, or examination of tumor markers for malignant lymphoma (e.g., sIL2-R) in the blood. Lymphoma may be diagnosed, for example, by the methods described in Nirmal (supra) or Voltin et al. (supra). The therapeutic effect of lymphoma may be evaluated, for example, by observing changes in lymphoma cell proliferation over time after drug administration. A state in which cell proliferation is inhibited includes a state in which the proliferation rate of test cells is significantly reduced compared to before drug treatment. The proliferation rate may be measured, for example, using absorbance as an index, or may be determined from image data. The therapeutic effect of lymphoma may be evaluated, for example, by observing a decrease in tumor mass after drug administration.In this case, a therapeutic effect may be determined if the tumor mass is significantly reduced compared to before administration of the drug or after administration of a negative control. The therapeutic effect on lymphoma may be measured, for example, using the amount of a lymphoma marker in the patient or a patient-derived sample as an index. In this case, a therapeutic effect may be determined if the marker amount is significantly reduced compared to before administration of the drug or after administration of the negative control. The tumor mass or marker amount after drug administration may be reduced to 0.9, 0.7, 0.5, 0.3, or 0.1 times or less compared to before administration or after administration of the negative control.

[0067] In the above (1) to (5), lymphoma includes MYC-positive, MYC-negative, EBV-positive, or EBV-negative lymphoma. By targeting MYC-positive or EBV-positive lymphoma, BCV, its pharmaceutically acceptable salt, or a solvate thereof can be highly effective in treating lymphoma that is either EBV-positive or EBV-negative, or both. By targeting MYC-positive lymphoma, BCV, its pharmaceutically acceptable salt, or a solvate thereof can be highly effective in treating lymphoma that is either EBV-positive or EBV-negative, or both.

[0068] In the above (1) to (5), "positive" includes positive gene expression. Positive may also include increased gene expression compared to healthy individuals or individuals previously confirmed to be negative. In the above (1) to (5), the expression level of MYC or EBV in a subject or a subject's lymphoma, or the positive / negative status of MYC or EBV, can be detected, for example, by collecting a subject's sample (e.g., lymphoma cells, plasma, whole blood, or tissue) and measuring the MYC or EBV gene expression in the sample. Gene expression can be measured, for example, by RT-PCR, DNA chip, or immunostaining. In this case, the sample collected from the subject may be compared with a sample collected from a healthy individual or an individual previously confirmed to be negative, and the sample collected from the subject may be determined to be MYC or EBV positive if the MYC or EBV gene expression is significantly elevated. EBV gene expression may also be measured using the expression of EBV-derived transcription products (e.g., EBER or EBNA1) as an indicator. Furthermore, the expression level of MYC or EBV in a subject or a subject's lymphoma, or the positivity / negativity of MYC or EBV, can be detected, for example, by collecting lymph nodes from the subject and immunostaining them for MYC or EBV. In this case, a sample collected from the subject may be compared with a sample collected from a healthy individual or an individual previously confirmed to be negative, and the sample collected from the subject may be determined to be MYC or EBV positive if the staining intensity of MYC or EBV is significantly increased. The increase in gene expression or staining intensity includes, for example, an increase of 1.5, 2, 3, 4, 5, 10, 20, or 50 times or more compared to the comparison subject, or an increase within a range of any two of these values. Furthermore, in detecting the positivity / negativity of MYC or EBV in a subject or a subject's lymphoma, for example, a certain percentage of cells in a sample immunostained for MYC or EBV may be determined to be MYC or EBV positive. This percentage may be, for example, 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more, or 100%, or may be within a range of any two of these values. When the expression level of EBV or MYC is used as an index, a high expression level of EBV or MYC in a subject specimen may include, for example, a high expression level of EBV or MYC relative to the expression level of a comparative specimen.This increase may be, for example, 1.5, 2, 3, 4, 5, 10, 20, or 50 times or more, or within a range of any two of these values. The comparison subject may be a sample previously confirmed not to have high EBV or MYC expression, or an EBV-negative or MYC-negative sample. Furthermore, for example, a high EBV or MYC expression level in a subject sample may include a relatively high level among a group of samples from lymphoma subjects. The group of samples may include, for example, samples from general patients with lymphoma. High expression or positivity may be assessed based on the common general technical knowledge of those skilled in the art, and may be performed, for example, using any one of the methods described in this paragraph. Furthermore, the expression level of MYC in a subject or a subject's lymphoma, or the positivity / negativity of MYC, can be detected, for example, by detecting MYC translocations using fluorescence in situ hybridization (FISH) (see, for example, Salam et al., J Cancer 2020; 11(1):190-198 or Epperla et al., Cancer. 2017 Nov 15;123(22):4411-4418). The expression level of EBV in a subject or a subject's lymphoma, or the positivity / negativity of EBV, can be detected, for example, by quantifying EBV DNA in plasma or whole blood using a commercially available DNA quantification kit (e.g., Accugene m-EBV (Abbott Japan G.K.)), or by detecting IgM antibodies against EBV capsid antigens in serum using a commercially available antibody detection kit (e.g., BioPlex EBV IgM Kit (Bio-Rad Laboratories, Inc.)). Detection of the expression level of MYC or EBV in a subject or in a subject's lymphoma, or positivity / negativity of MYC or EBV, may be performed, for example, in vitro, ex vivo, or in vivo.

[0069] In the above (1) to (5), lymphomas include, for example, double-hit or triple-hit lymphomas. Double-hit lymphomas include, for example, lymphomas with MYC and BCL2 gene rearrangements. Triple-hit lymphomas include, for example, lymphomas with MYC, BCL2, and BCL6 gene rearrangements. Double-hit or triple-hit lymphomas can be detected by detecting translocations using, for example, FISH (see, for example, Salam et al. (supra) or Epperla et al. (supra)). Lymphomas include, for example, high-grade lymphomas.

[0070] In the above (1) to (5), MYC includes proteins known as transcriptional regulators. Herein, MYC, Myc, and c-Myc can be used interchangeably or with substantially the same meaning. The primary accession number for MYC listed in UniProt is, for example, P01106. EBV is an abbreviation for Epstein-Barr virus. EBV is known as a virus with double-stranded DNA (see, for example, Rivailler et al., J. Virol. 2002 Dec; 76(23): 12055-12068 or Correia et al., J. Virol. 2018 Nov 15; 92(22): e01132-18).

[0071] In the above (1) to (5), the chemotherapeutic agent is not particularly limited and includes, for example, an anticancer agent. Anticancer agents include, for example, microtubule inhibitors, DNA synthesis inhibitors, growth factor inhibitors, tyrosine kinase inhibitors, cytotoxic substances, immune checkpoint inhibitors, or other anticancer agents. Microtubule inhibitors include, for example, vinca alkaloids or taxanes. Vinca alkaloids include, for example, vincristine, vinblastine, vindesine, vinorelbine, or eribulin. Taxanes include, for example, paclitaxel or docetaxel. DNA synthesis inhibitors include, for example, antimetabolites, topoisomerase inhibitors, platinum compounds, antitumor antibiotics, or alkylating agents. Antimetabolites include, for example, pemetrexed, 5-fluorouracil, S-1, gemcitabine, or capecitabine. Examples of topoisomerase inhibitors include irinotecan, nogitecan, etoposide, and zobuzoxacin. Examples of platinum-based agents include cisplatin, oxaliplatin, nedaplatin, and carboplatin. Examples of antitumor antibiotics include anthracyclines (e.g., doxorubicin, liposomal doxorubicin, daunorubicin, epirubicin, idarubicin, aclarubicin, amrubicin, mitoxatrone, and pirarubicin), mitomycin C, actinomycin D, bleomycin, beplomycin, and zinostatin stimulant. Examples of alkylating agents include bendamustine, cyclophosphamide, dacarbacine, and ifosfamide. Examples of growth factor inhibitors include inhibitors of EGF, VEGF, FGF, and IGF. Examples of growth factor inhibitors include bevacizumab, cetuximab, and panitumumab. Examples of tyrosine kinase inhibitors include gefitinib and erlotinib. Examples of cytotoxic substances include saporin, emtansine, deruxtecan, and vedotin. Examples of immune checkpoint inhibitors include drugs that bind to immune checkpoint molecules or their ligands to inhibit the transmission of immunosuppressive signals, thereby relieving the suppression of T cell activation by immune checkpoint molecules.Immune checkpoint inhibitors include, for example, anti-CTLA-4 antibodies (e.g., ipilimumab), anti-PD-1 antibodies (e.g., nivolumab or pembrolizumab), or anti-PD-L1 antibodies (e.g., atezolizumab or avelumab). Other anticancer drugs include, for example, L-asparaginase. Forms of chemotherapeutic agents include, for example, low molecular weight compounds or high molecular weight compounds. Chemotherapeutic agents include salts of any one or more of the compounds shown herein (including the salts shown below).

[0072] In the above (1) to (5), the salt is not particularly limited and includes, for example, inorganic salts or organic salts (see, for example, "Bharate et al., Drug Discov Today. 2021 Feb;26(2):384-398" or "Berge et al., J Pharm Sci. 1977 Jan;66(1):1-19."). Salts include, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Metal salts include, for example, alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, and the like. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, and the like. Salts with inorganic acids include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with organic acids include, for example, salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, mesylic acid, tosylic acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Salts with basic amino acids include, for example, salts with arginine, lysine, ornithine, etc. Salts with acidic amino acids include, for example, salts with aspartic acid, glutamic acid, etc. Salts include pharmaceutically acceptable salts. In one embodiment of the present invention, pharmaceutically acceptable includes forms having reasonable benefits for pharmaceutical use. In one embodiment of the present invention, a form of the compound or a salt thereof includes a solvate form thereof. In the above (1) to (5), solvates include forms of compounds formed by a solute and a solvent (see, for example, Healy et al., Adv Drug Deliv Rev. 2017 Aug 1;117:25-46.) Solvates include, but are not limited to, hydrates (e.g., monohydrate, dihydrate, trihydrate, etc.) or organic solvent solvates (e.g., solvates with alcohols (e.g., methanol, ethanol, propanol, etc.), acetone, dimethylformamide, or ethyl acetate, etc.).Solvents include those that are capable of substantially maintaining the biological activity of the solute after forming the solvate. Solvates include pharmaceutically acceptable solvates.

[0073] In the above (1) to (5), "treatment" includes the possibility of exerting a symptom-improving, suppressive, recurrence-suppressive, or preventive effect on a patient's disease or one or more symptoms associated with the disease. Furthermore, "treatment" also includes, for example, treatments that suppress the proliferation of lymphoma cells in a patient, reduce lymphoma markers, suppress lymph node swelling, and suppress lymph node recurrence. In the above (1) to (5), the pharmaceutical composition may be prepared by any method known in the technical field of pharmaceuticals, for example, by mixing an active ingredient with one or more pharmaceutically acceptable carriers. Furthermore, the pharmaceutical composition may be used in any form, as long as it is used for treatment. It may contain the active ingredient alone or a mixture of the active ingredient and any other ingredient. The form of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The content of the carrier may be, for example, a pharmaceutically effective amount. The effective amount may be, for example, an amount sufficient for pharmaceutical stability or delivery of the active ingredient. For example, a buffer solution is effective for stabilizing the active ingredient in a vial. The pharmaceutical composition may also contain a stabilizer (e.g., mannitol), a buffer (e.g., arginine), or a pH adjuster (e.g., NaOH). The dosage, administration interval, administration method, and administration route are not particularly limited and can be appropriately selected depending on the patient's age and weight, symptoms, target organ, etc. Furthermore, the pharmaceutical composition preferably contains a therapeutically effective amount, or an effective amount that exerts the desired effect, of the active ingredient. In one embodiment of the present invention, a therapeutically effective amount includes the amount necessary to clinically observe improvement in symptoms in the patient. In one embodiment of the present invention, "pharmaceutically acceptable" includes a state that is suitable for use within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. There are no particular limitations on the components other than BCV in the pharmaceutical composition, as long as they do not impair the effects of the present invention, and they can be appropriately selected depending on the purpose. In the above (1) to (5), the treatment may not include, for example, concomitant use with probenecid, a uricosuric agent, or a drug that suppresses side effects, if specifically specified. Side effects include, for example, diarrhea or nephrotoxicity.

[0074] In the above (1) to (5), the route of administration of BCV, a pharmaceutically acceptable salt thereof, or a solvate thereof, or a pharmaceutical composition thereof to a subject is preferably one that is effective for the treatment, and may be, for example, oral, intravenous, subcutaneous, intramuscular, or intraperitoneal. The dosage form is preferably one that is effective for the treatment, and may be, for example, a solid preparation (e.g., tablet), a liquid preparation (e.g., oral suspension), or an injection (e.g., intravenous injection).

[0075] In the above (1) to (5), the dosage, administration interval, and administration method of BCV, its pharmaceutically acceptable salt, or solvate thereof, or pharmaceutical composition thereof to a subject can be appropriately selected depending on the patient's age, body weight, symptoms, target organ, etc. The dosage may be, for example, 0.01 to 200 mg / kg body weight per administration. The administration interval may be, for example, once or twice every 1 to 28 days or every 1 to 4 weeks. More specifically, this includes intravenous injection of 20 to 80 mg / day. It may also include intravenous injection of 20 to 80 mg / day twice weekly. Furthermore, during combination therapy with BCV and a chemotherapeutic agent (e.g., gemcitabine, etoposide, or an immune checkpoint inhibitor), the BCV dosage may include intravenous injection of 10 to 40 mg or 10 to 20 mg / day. It may also include intravenous injection of 10 to 40 mg or 10 to 20 mg / day twice weekly. The preferred human dosage is 10 to 80 mg / day twice weekly, preferably by intravenous injection. This can minimize side effects while demonstrating excellent therapeutic efficacy against lymphoma. The range of 10 to 80 mg indicated in this paragraph may be, for example, 10, 20, 30, 40, 50, 60, 70, or 80 mg, or may be within a range of any two of these.

[0076] In one embodiment of the present invention, "significantly" may mean, for example, that a statistically significant difference is evaluated using a Student's t-test (one-tailed or two-tailed) and p<0.05 or p<0.01, or that a substantial difference is present.

[0077] All publications cited herein are incorporated by reference in their entirety. As used herein, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "or." As used herein, when it is stated that "within a range of two values," the range also includes the two values ​​themselves. As used herein, "A to B" includes A and B. As used herein, "having" in relation to a disease includes being affected.

[0078] Although the embodiments of the present invention have been described above, these are merely examples of forms that may be included in the present invention, and the present invention is not limited to these, and various configurations other than those described above may also be adopted. Furthermore, the present invention may be adopted by combining or independently adopting each of the configurations or features described in the above embodiments.

[0079] The present invention will be further explained below with reference to examples, but is not limited to these.

[0080] Example 1: Growth inhibition of lymphoma cells Cell viability was measured using the Promega CellTiter-Glo® 2.0 Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol: 2 × 10 lymphoma cells in 100 μL of medium. 3Cells were seeded into 96-well plates at a concentration of 0.01%. Each well was treated with various concentrations of drug (BCV). At each time point, Promega CellTiter-Glo® 2.0 Cell Viability Assay reagent was added to the wells. After 10 minutes of incubation at room temperature, absorbance at 450 nm was measured using a Tecan M200 Infinite 96-well plate reader and IControl software 1.6 (Tecan, Männedorf, Switzerland). Cell viability was assessed as a percentage of control absorbance. Growth inhibitory effects were analyzed by generating dose-response curves plotting the percentage of viable cells against drug concentration, and their IC50 values ​​were estimated using GraphPad Prism version 8.0.2 (GraphPad Software). All reactions were performed in triplicate. EBV-positive / negative cells were also confirmed by detecting EBV-derived transcripts (e.g., EBER, EBNA1).

[0081] The results are shown in Figure 1. BCV inhibited the proliferation of NK / T lymphoma cells. Regarding this cell proliferation inhibitory effect, the IC50 values ​​were significantly lower in EBV-positive NK / T lymphoma cells (KAI-3, NK-S1) than in EBV-negative NK / T lymphoma cells (KHYG-1).

[0082] Example 2: Comparison with antiviral drugs The experimental procedures were the same as in Example 1, except that the drugs used were BCV, acyclovir, ganciclovir, adefovir, foscarnet, and penciclovir.

[0083] The results are shown in Figure 2. BCV exhibited a significantly superior cell growth inhibitory effect against EBV-positive NK / T lymphoma cells (NK-S1) compared with other antiviral drugs (acyclovir, ganciclovir, adefovir, foscarnet, and penciclovir).

[0084] Example 3: In vivo drug treatment For in vivo drug treatment with BCV, 6-week-old female NSG mice were administered 0.5x10 6 The mice were inoculated with NK-S1 cells and intraperitoneally administered BCV or a control vehicle at a dose of 40 mg / kg twice a week.3 Tumor measurements were recorded twice weekly until tumor size reached 100 mg / kg. Mice were euthanized according to IACUC guidelines. Tumor sizes in the experimental and control groups (n = 8 per group) were averaged at each time point (days 1, 5, 8, 12, and 15) and statistically compared. Signs of toxicity, including diarrhea and weight loss, were monitored throughout the experiment.

[0085] The results are shown in Figures 3A-D. Administration of BCV significantly suppressed the growth of EBV-positive NK / T lymphoma. BCV was shown to have a high therapeutic effect in vivo. Furthermore, the animals were in good health and no diarrhea was observed. This suggests that side effect management can be omitted or simplified.

[0086] Example 4: Reduction of EBNA1 and LMP1 expression After treating NK-S1 and KAI-3 cell lines with 0.1 and 1 μg / mL BCV for 72 hours, whole cell lysates were diluted with 4-15% Mini-PROTEAN TM TGX Stain-Free TM Protein samples were separated by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA) using Protein Gel and transferred to a 0.2 μm PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). After blocking, the membrane was gently shaken overnight at 4°C in a solution containing 5% nonfat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) or 5% bovine serum albumin (Sigma-Aldrich, Darmstadt, Germany), TBST solution (50 mM Tris / HCl pH 7.4, 150 mM NaCl, 0.1% Tween-20), and primary antibodies (anti-EBNA1 antibody (Santa Cruz) or anti-LMP1 antibody (Dako)). Exposure to the appropriate HRP-conjugated anti-mouse antibody (Cytiva, Washington, DC, USA) was performed for 1 h, followed by final chemiluminescence detection using the SuperSignal Substrate Western Blotting Kit (Thermo Fisher Scientific, MA, USA). ChemiDoc TM XRS+ (Image LabTM Imaging was performed using a system equipped with software (Bio-Rad Laboratories, Hercules, CA, USA).

[0087] The results are shown in Figure 4. BCV treatment reduced the expression levels of EBNA1 and LMP1 in lymphoma cells.

[0088] Example 5: Reduction of Myc Expression. Whole transcript sequencing was performed on NK-S1 and KAI-3 cell lines treated with BCV. Gene set enrichment analysis (GSEA) was performed using the Hallmark gene set from the Molecular Signatures Database (MSigDB). Gene sets were considered significantly enriched if the False Discovery Rate (FDR) q-value of the normalized enrichment score (NES) was less than 0.05. Quantitative PCR was performed using Maxima SYBR Green / ROX qPCR Master Mix (Thermo Scientific, USA). The ΔCt of a gene's mRNA expression was calculated as the difference from the Ct value of the internal control, GAPDH. ΔΔCt was calculated by subtracting the ΔCt value of the treated group from the ΔCt value of the untreated control group. The expression ratio was calculated using the following formula: -ΔΔCt was calculated using

[0089] The results are shown in Figure 5A–C. BCV significantly reduced Myc expression and the expression of genes controlled by MYC, while the expression of genes in the STING pathway increased.

[0090] Example 6: Induction of Immunogenic Cell Death. NK-S1 and KAI-3 cell lines were treated with BCV (0.1 μg / mL or 1 μg / mL) or DMSO alone for 72 hours. For calreticulin staining, cells were incubated with calreticulin antibody (1:100, room temperature for 30 minutes) (#ab92516, Abcam), washed, resuspended in PBS, and analyzed using a cell analyzer (BD LSR Fortessa, BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo version 10.8.0 (BD Biosciences, San Jose, CA, USA). Extracellular levels of HMGB1 upon BCV treatment were measured using the Lumit HMGB1 immunoassay (Promega, Madison, WI, USA).

[0091] The results are shown in Figure 6. Typical phenotypes reflecting immunogenic cell death, such as an increase in calreticulin-positive cells and HMGB1 release, were clearly elevated by BCV treatment.

[0092] Example 7: Combination Treatment. The NK-S1 cell line was treated with selected doses of etoposide, gemcitabine, or gamma irradiation, with or without BCV (0.1 μg / mL). Cell viability was quantified using the Promega CellTiter-Glo® 2.0 Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. The Combination Index (CI) was calculated using Chou-Talalay median-effect analysis to determine synergy, additivity, or antagonism. CI values ​​<1 or >1 indicate synergy or antagonism, respectively. CI values ​​close to 1 (i.e., between 0.9 and 1.1) were considered additive.

[0093] The results are shown in Figures 7A-C. Combined treatment of BCV with etoposide or gemcitabine significantly enhanced the effect compared with treatment with either drug alone. The combination index indicated a synergistic effect.

[0094] Example 8: Growth Inhibition of MYC-Positive Lymphoma Cells. Various B-cell lymphoma cell lines were treated with various concentrations of BCV for 96 hours. Cell viability was quantified using the Promega CellTiter-Glo® 2.0 Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. The presence or absence of EBV and molecular information were referenced from public databases. The cell lines shown in Figure 8 have been reported to be classified into the following types: Raji and Daudi: Burkitt lymphoma (BL) cell lines; VAL and DB: diffuse large B-cell lymphoma (DLBCL) cell lines; and BJAB: BL or DLBCL cell lines. All are B-lymphomas with MYC translocation or amplification. Raji, Daudi, and VAL are EBV-positive, while BJAB and DB are EBV-negative cell lines. VAL is a triple-hit lymphoma, and DB is a double-hit lymphoma.

[0095] The results are shown in Figure 8. BCV exhibited a clear cell growth inhibitory effect on MYC-positive lymphoma cells. It also showed a high growth inhibitory effect on both EBV-positive and -negative cells. In Example 5, BCV reduced Myc expression, and in Example 8, BCV inhibited the growth of MYC-positive lymphoma cells. These results demonstrated that BCV is particularly suitable for the treatment of MYC-positive lymphoma.

[0096] Example 9: Growth Inhibition of 11 Lymphoma Cell Lines The effect of BCV on cell viability was examined for 11 NK / T lymphoma cell lines (KAI-3, NK-S1, NK-92, KHYG-1, NK-YS, MEC-04, SNK-1, SNK-6, YT, HANK-1, and SNT-8). Cell viability was quantified using the Promega CellTiter-Glo® 2.0 Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol. Specifically, cells were cultured at 2 × 10 in 100 μL of medium. 3Cells were seeded into 96-well plates at a concentration of 0.01% and drugs were added at various concentrations. After each time point, Promega CellTiter-Glo® 2.0 Cell Viability Assay reagent was added to the wells, and after 10 minutes of incubation at room temperature, absorbance at 480 nm was measured using a Tecan M200 Infinite 96-well plate reader and iConrol software 1.6 (Tecan, Männedorf, Switzerland). Cell viability was calculated as a percentage of control absorbance. Growth inhibitory effects were analyzed by generating dose-response curves as a plot of the percentage of viable cells against drug concentration, and their IC50 values ​​were estimated using GraphPad Prism version 8.0.2 (GraphPad Software). All reactions were performed in triplicate.

[0097] The results are shown in Figure 9. Four cell lines (KAI-3, NK-S1, NK-92, and KHYG-1) showed lower IC50 values ​​and higher susceptibility to BCV than the other cell lines.

[0098] Example 10: Whole-transcriptome sequencing of 11 lymphoma cell lines. Whole-transcriptome sequencing was performed on the same 11 NK / T lymphoma cell lines as in Example 9. Gene set enrichment analysis (GSEA) was performed using the Molecular Signatures Database (MSigDB) Hallmark gene set to compare the top four BCV-susceptible cell lines (KAI-3, NK-S1, NK-92, and KHYG-1) with the other seven cell lines. Gene sets were considered significantly variable if the False Discovery Rate (FDR) q value of the normalized enrichment score (NES) was less than 0.05.

[0099] The results are shown in Figures 10A and 10B. The significantly elevated MYC TARGETS V2 in the figure represents a group of genes regulated by MYC. Analysis revealed that four susceptible cell lines (KAI-3, NK-S1, NK-92, and KHYG-1) had significantly higher gene expression than the other seven cell lines, with the highest expression observed in the group of genes regulated by MYC. This indicates that the higher the MYC expression, the greater the antitumor effect of BCV.

[0100] Example 11: Impact on Prognosis Progression-free survival and overall survival were investigated for a cohort of human NK / T-cell lymphoma patients (n=36). Furthermore, based on the results of genetic analysis, the patient cohort was divided into Group A (n=19) and Group B (n=17). Enrolled patients were those who had not received BCV treatment. The results are shown in Figures 11A and 11B. Comparing Group A and Group B, Group A tended to have a good prognosis, while Group B tended to have a poor prognosis. Statistical analysis was performed using MedCalc for Windows, version 18.2.1 (MedCalc Software).

[0101] Furthermore, based on the results obtained from whole-transcriptome sequencing in the above human patient cohort (n = 36), we performed gene set enrichment analysis (GSEA) using the Molecular Signatures Database (MSigDB) Hallmark gene sets to compare favorable and unfavorable prognosis groups. Gene sets were considered significantly altered if the False Discovery Rate (FDR) q value of the normalized enrichment score (NES) was less than 0.05.

[0102] The results are shown in Figure 12. In the figure, MYC TARGETS V1 and MYC TARGETS V2, which showed significant increases, are gene groups regulated by MYC. Analysis revealed that the gene group regulated by MYC was significantly more highly expressed in the poor prognosis group than in the good prognosis group. As described in Example 5, BCV significantly reduced Myc expression and also significantly reduced the expression of genes controlled by MYC. Therefore, administration of BCV is thought to have the effect of improving prognosis.

[0103] The present invention has been described above based on the embodiments. However, these embodiments are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims

1. A pharmaceutical composition for treating lymphoma, comprising brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof.

2. The pharmaceutical composition of claim 1, comprising brincidofovir.

3. The pharmaceutical composition according to claim 1 or 2, wherein the lymphoma is malignant lymphoma.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the lymphoma is NK / T cell lymphoma or B cell lymphoma.

5. A pharmaceutical composition according to any one of claims 1 to 4, wherein the lymphoma is NK / T cell lymphoma.

6. A pharmaceutical composition according to any one of claims 1 to 5, which reduces MYC expression in lymphoma cells of the subject of said treatment.

7. A pharmaceutical composition according to any one of claims 1 to 6, wherein said treatment comprises identifying an EBV-positive subject as a target for treatment.

8. A pharmaceutical composition according to any one of claims 1 to 7, wherein the lymphoma is EBV-positive lymphoma.

9. A pharmaceutical composition according to any one of claims 1 to 8, wherein the treatment comprises detecting the expression level of EBV in a subject's specimen.

10. A pharmaceutical composition according to any one of claims 1 to 9, wherein the treatment comprises identifying a MYC-positive subject as a treatment target.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the lymphoma is a MYC-positive lymphoma.

12. A pharmaceutical composition according to any one of claims 1 to 11, wherein the treatment comprises detecting the expression level of MYC in a subject's sample.

13. A pharmaceutical composition according to any one of claims 1 to 12, wherein the treatment comprises identifying an EBV-positive and MYC-positive subject as a treatment target.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the lymphoma is EBV-positive and MYC-positive.

15. A pharmaceutical composition according to any one of claims 1 to 14, wherein the treatment comprises detecting the expression levels of EBV and MYC in a subject's sample.

16. A pharmaceutical composition according to any one of claims 1 to 15, which improves prognosis.

17. A pharmaceutical composition according to any one of claims 1 to 16, which inhibits the proliferation of lymphoma cells.

18. The pharmaceutical composition according to any one of claims 1 to 17, for use in combination therapy of brincidofovir, a pharmaceutically acceptable salt thereof, or a solvate thereof with a chemotherapeutic agent.

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