Epstein-Barr virus replication inhibitors

Abstract

Provided is a novel antiviral activator capable of inhibiting replication of Epstein-Barr virus. Also provided is a composition for inhibiting the replication of Epstein-Barr virus, the composition comprising auranofin, fenofibrate, or a combination thereof.

Description

[Technical Field]

[0001] The present disclosure relates to gammaherpesviruses, and in particular to Epstein-Barr virus (EBV) in humans. [Background technology]

[0002] EBV infects humans and is associated with a variety of diseases, and the infection process involves multiple stages: a primary infection, a latent infection in which the virus remains dormant in the host after primary infection but does not necessarily cause disease, even though it expresses only a portion of its genes, and a reactivation stage in which the virus is triggered by factors such as a weakened immune system.

[0003] EBV infection is not uncommon; in fact, the majority of adults are infected with EBV. EBV is known to be transmitted from person to person via saliva. Primary infection often occurs during infancy and is almost always asymptomatic, but occasionally (especially if primary infection occurs after adolescence) can cause a disease called infectious mononucleosis. It is thought that there is an incubation period of approximately 4 to 6 weeks between actual viral infection and the onset of disease. Infectious mononucleosis is characterized by fever, redness of the throat, and splenic swelling, and approximately 70% of patients also develop hepatitis. While most cases of infectious mononucleosis resolve spontaneously, in rare cases, hepatomegaly, massive splenomegaly, and splenic rupture can occur. Complications include central nervous system symptoms such as aseptic meningitis. At the cellular level, EBV primarily infects lymphocytes, particularly B cells, and can progress to lymphoproliferative disorders and hemophagocytic syndrome, so caution is required.

[0004] Individuals who experience primary EBV infection remain carriers of the virus for life. Latent EBV infection in adults can be reactivated by factors such as immunosuppression, aging, and stress. When EBV is reactivated, the virus particles and the viral genome within them begin replicating again, potentially causing host cell lysis. EBV reactivation has been linked to a variety of diseases, including neurological disorders such as multiple sclerosis and Alzheimer's disease, autoimmune diseases such as rheumatoid arthritis, SLE, and Graves' disease, and even the aftereffects of COVID-19 infection. Furthermore, EBV-associated lymphoproliferative disorders, which can occur with immunosuppressant administration during transplantation, methotrexate treatment in rheumatoid arthritis patients, and biologics in inflammatory bowel disease patients, are also a clinical problem.

[0005] However, no EBV-specific treatment has been established, and the development of new anti-EBV drugs is highly desired. Because the main pathology of EBV-associated diseases is a strong inflammatory response of the host to EBV-infected cells, treatment with anti-inflammatory steroids is the primary method, but this is only a symptomatic treatment and does not address the root cause of the virus itself.

[0006] EBV is a species-specific virus that infects only humans. Therefore, even now, more than half a century after the discovery of EBV, experimental analysis of EBV itself in individual animals or animal cells is difficult, hindering the analysis of events immediately after primary infection and the promotion of therapeutic drug evaluation. However, MHV68, a mouse virus belonging to the same gamma herpes subfamily, exhibits a mouse infection process highly similar to that occurring in human EBV infection, and the MHV68 infection system has been accepted worldwide as an EBV infection model based on knowledge accumulated over many years (Non-Patent Document 1).

[0007] Non-Patent Document 2 describes the inhibition of EBV replication by the drug dipyridamole in a reactivation experiment using a human cell line harboring latently infected EBV. Prior to Non-Patent Document 2, dipyridamole was known to be an inhibitor of nucleoside transport, to be an antiviral agent against various types of viruses, and to exert its antiviral activity via inhibition of viral nucleic acid replication (Non-Patent Documents 3-5). As Non-Patent Document 5 calls it a "modified purine" or "purine analog," dipyridamole is structurally characterized by a fused pyrimidine ring similar to the purine in nucleosides. This structure is thought to be related to dipyridamole's mechanism of action, which is the inhibition of nucleoside transport and nucleic acid replication. As mentioned in Non-Patent Document 2, several other compounds with structures similar to nucleotide building blocks, including nucleoside analogs, nucleotide analogs, and pyrophosphate analogs, are also known to be capable of inhibiting EBV replication. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Mistrikova et al., Acta Virologica, 2020;64(2):167-176 [Non-patent document 2] Thome et al., Antiviral Research 172 (2019) 104615 [Non-patent document 3] Tenser et al., Antimicrob Agents Chemother., 2001, 45(12): 3657-3659 [Non-patent document 4] Tonew et al., Chemotherapy. 1977, 23(3):149-58 [Non-patent document 5] Fata-Hartley et al., J Virol. 2005, 79(17): 11062-11070 Summary of the Invention [Problem to be solved by the invention]

[0009] In the field of anti-EBV treatment, where options are currently limited, the development of new drugs is highly desirable. The objective of the present disclosure is to provide novel antiviral active agents capable of inhibiting EBV replication. [Means for solving the problem]

[0010] The present inventors have discovered that known drugs called auranofin and fenofibrate can inhibit or suppress the replication of human EBV and its mouse model, MHV68. Embodiments of the present invention are based on this discovery. Auranofin and fenofibrate are known as anti-inflammatory drugs for rheumatoid arthritis patients and anti-hyperlipidemic drugs, respectively. Recently, it has been reported that auranofin and fenofibrate can inhibit the replication of the novel coronavirus SARS-CoV2 (Rothan et al., Virology 547 (2020) 7-11; Ehrlich et al., eLife 2023;12:e79946). However, it was completely unknown that these drugs could inhibit EBV replication.

[0011] The present disclosure provides the following embodiments. [1] A composition for inhibiting Epstein-Barr virus replication, comprising auranofin, fenofibrate, or a combination thereof. [2] The composition described in [1], which is a pharmaceutical composition for administration to a subject in need of inhibiting Epstein-Barr virus replication. [3] The composition described in [2], wherein the subject has a primary Epstein-Barr virus infection. [4] The composition described in [2], wherein the subject has latent infection or reactivation of Epstein-Barr virus. [5] The composition according to any one of [2] to [4], wherein the subject is an Epstein-Barr virus-infected patient having a disease selected from the group consisting of infectious mononucleosis, chronic active Epstein-Barr virus infection, lymphoproliferative disease, hemophagocytic syndrome, and autoimmune disease. [6] A pharmaceutical composition comprising auranofin, fenofibrate, or a combination thereof for the treatment or prevention of a disease caused by Epstein-Barr virus infection, wherein the disease is infectious mononucleosis, chronic active Epstein-Barr virus infection, lymphoproliferative disease, hemophagocytic syndrome, or an autoimmune disease. [Brief explanation of the drawings]

[0012] [Figure 1] The line graph shows the cell viability of 3T12 cells (left) or MLE12 cells (right) cultured in the presence of auranofin. The bar graph shows the viral load, i.e., the number of viral copies detected per ml of culture supernatant, in cells cultured in the presence of the corresponding concentrations of auranofin. [Figure 2] The line graph shows cell viability in 3T12 cells (left) or MLE12 cells (right) cultured in the presence of fenofibrate, and the bar graph shows the viral load (i.e., the number of viral copies detected per ml of culture supernatant) in cells cultured in the presence of the corresponding concentrations of fenofibrate. [Figure 3]The line graph shows cell viability in 3T12 cells (left) or MLE12 cells (right) cultured in the presence of dipyridamole, a known EBV replication inhibitor, and the bar graph shows the viral load, i.e., the number of viral copies detected per ml of culture supernatant, in cells cultured in the presence of corresponding concentrations of dipyridamole. [Figure 4] Figure 4 shows the EBV genome copy number (a), BART gene relative expression (b), BZLF1 and BRLF1 gene relative expression (c), and BLLF1 gene relative expression (d) in EBV-infected AGS cell lines treated with solvent control (white) or auranofin (black). DETAILED DESCRIPTION OF THE INVENTION

[0013] In one aspect, the present disclosure provides a composition for inhibiting EBV replication, comprising auranofin, fenofibrate, or a combination thereof. Auranofin and fenofibrate are known drugs approved for separate pharmaceutical uses, and basic knowledge has already been accumulated regarding their safety, dosage forms and delivery routes, pharmacokinetics, etc. Embodiments of the present disclosure can also be described as an EBV replication inhibitor comprising auranofin, fenofibrate, or a combination thereof; auranofin, fenofibrate, or a combination thereof for use in inhibiting EBV replication or treating or preventing an exemplary disease; or a method for inhibiting EBV replication or treating or preventing an exemplary disease using auranofin, fenofibrate, or a combination thereof. Corresponding to these embodiments, there is also provided use of auranofin, fenofibrate, or a combination thereof in the manufacture of a medicament for inhibiting EBV replication or treating or preventing an exemplary disease.

[0014] The structures of auranofin (left) and fenofibrate (right) are shown below. Both auranofin and fenofibrate can be said to be new types of EBV replication inhibitors that lack clear structural similarity to nucleotide components, unlike known EBV replication inhibitors, and multiple embodiments of the present invention have corresponding technical features in this regard.

[0015] [ka]

[0016] In the present disclosure, "replication" of EBV refers to the active increase in the copy number of the viral genome and viral particles by EBV, as understood by those skilled in the art. EBV replication can typically result in host cell lysis. EBV copy number can be quantified based on the amount of EBV genomic DNA.

[0017] The composition of this embodiment can be delivered to cells or tissues, for example, in vitro or ex vivo. Alternatively, the composition of this embodiment can be provided as a pharmaceutical composition for administration to a subject who would benefit from, i.e., who is in need of, inhibiting EBV replication. A "subject" may also be referred to as a "patient." In vivo, the composition can be administered to a subject via an appropriate delivery route known to those skilled in the art for auranofin and fenofibrate. For example, oral or intravenous administration is preferred. Other administration routes, including inhalation administration, topical administration, etc., are also contemplated. EBV replication in vivo is known to occur primarily in epithelial cells (particularly pharyngeal epithelial cells) and / or B cells, and a person skilled in the art can select an appropriate administration route based on common knowledge.

[0018] The composition of this embodiment can be used to treat or prevent EBV-associated disorders or diseases. That is, the composition of this embodiment can be used to treat or prevent EBV replication and disorders or diseases caused thereby. In some embodiments, the subject is a subject with primary EBV infection. For example, the composition can be used to treat infectious mononucleosis, and embodiments in which the composition is used to prevent infectious mononucleosis are also contemplated. In some embodiments, the subject is a subject with latent EBV infection or reactivation. Numerous EBV-associated disorders or diseases in which EBV reactivation is thought to contribute to the pathogenesis are known, and the composition can be used to treat or prevent these disorders or diseases. For example, as described above, it is known that EBV reactivation can occur in conjunction with the administration of immunosuppressants. Therefore, the composition of this embodiment can be administered in the setting of such immunosuppressant administration, i.e., to subjects receiving immunosuppressants, to suppress EBV replication or prevent EBV-associated disorders or diseases. Embodiments in which the composition is administered to reduce EBV replication in subjects in whom EBV replication has already begun or to treat EBV-associated disorders or diseases are also contemplated.

[0019] In some embodiments, the subject may be an EBV-infected patient with a disease selected from the group consisting of infectious mononucleosis, chronic active EBV infection, lymphoproliferative disease, hemophagocytic syndrome, and autoimmune disease. For example, the subject may be an EBV-infected patient with primary EBV infection and infectious mononucleosis, or an EBV-infected patient with EBV reactivation and chronic active EBV infection, lymphoproliferative disease, hemophagocytic syndrome, or autoimmune disease. The autoimmune disease may be, for example, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, etc. The subject may also be a patient with a neurological disease such as multiple sclerosis or Alzheimer's disease, or a patient with sequelae of COVID-19 infection. In certain embodiments, the subject (particularly a subject receiving auranofin) may not be a patient with rheumatoid arthritis. In certain embodiments, the subject (particularly a subject receiving fenofibrate) may not be a patient with dyslipidemia, hyperlipidemia, hypertriglyceridemia, or hypercholesterolemia. In certain embodiments, the subject may not be a patient with SARS-CoV2 infection. Inhibiting EBV replication in the patient can result in treating or preventing a disease identified as being caused by EBV replication. Thus, provided is a pharmaceutical composition comprising auranofin, fenofibrate, or a combination thereof for treating or preventing a disease caused by EBV infection, wherein the disease is infectious mononucleosis, chronic active Epstein-Barr virus infection, lymphoproliferative disorder, hemophagocytic syndrome, or an autoimmune disease.

[0020] The composition of this embodiment may contain a pharmaceutically acceptable carrier or excipient in addition to the active ingredients auranofin and / or fenofibrate. Thus, the manufacture of a pharmaceutical of this embodiment may involve combining auranofin and / or fenofibrate with a pharmaceutically acceptable carrier or excipient. Specific examples of such carriers or excipients include, but are not limited to, water, propylene glycol, ethanol, vegetable oil, mineral oil, dimethyl sulfoxide, glycerin, lactose, sucrose, mannitol, starch, dextrin, cellulose, cellulose ether, polyethylene glycol, polyvinylpyrrolidone, magnesium stearate, talc, and any combination thereof. The composition or pharmaceutical composition for inhibiting EBV replication of this embodiment may be provided in the form of a liquid formulation. In another embodiment, the composition or pharmaceutical composition for inhibiting EBV replication may be provided as a solid formulation such as a tablet, pill, powder, or capsule, or as a semi-solid formulation.

[0021] Hereinafter, the embodiments will be described in more detail with reference to examples, but the examples are merely illustrative and the embodiments of the invention are not limited to these examples. [Example]

[0022] Example 1 We have found that auranofin and fenofibrate each have inhibitory activity against EBV replication. The following experimental results are presented using the murine MHV68 infection system, an established model of primary EBV infection, and comparing them with dipyridamole, a known EBV replication inhibitor. These drugs also exhibit inhibitory activity against EBV reactivation.

[0023] Mouse 3T12 or MLE12 cell lines were cultured in multiwell culture plates using standard methods. These cell lines were treated with auranofin, fenofibrate, or dipyridamole at different concentrations. Six days after treatment, cell viability was measured using the well-known WST-8 method. WST-8 is a tetrazolium salt that is converted by dehydrogenases in viable cells into a formazan compound that absorbs at specific wavelengths.

[0024] In the corresponding experiments, each of the above cell lines was 2 Mice were infected with pfu of virus and simultaneously administered the above-mentioned concentrations of auranofin, fenofibrate, or dipyridamole. Three days after administration, the viral genome copy numbers in the culture supernatant were measured by quantitative polymerase chain reaction (qPCR).

[0025] For example, in Figure 1, the line graphs show the cell viability of 3T12 cells (left) or MLE12 cells (right) cultured in the presence of auranofin at the concentrations shown at the bottom. For reference, a horizontal line corresponding to 80% cell viability is shown. The bar graphs show the viral load, i.e., the number of viral copies detected per ml of culture supernatant, in cells cultured in the presence of the corresponding concentrations of auranofin. These data are shown as the average of measurements performed in triplicate in quadruplicate wells. Error bars indicate standard deviation, and * indicates statistical significance (P<0.05).

[0026] Figure 2 shows an experiment similar to that shown in Figure 1, except that fenofibrate was administered instead of auranofin. Figure 3 shows an experiment similar to that shown in Figure 1, except that dipyridamole was administered instead of auranofin. Dipyridamole is a drug whose inhibitory activity against EBV replication was shown by Non-Patent Document 2.

[0027] As illustrated in these figures, auranofin and fenofibrate each had viral replication inhibitory activity at least comparable to or superior to that of dipyridamole, and these viral replication inhibitory activities were achieved at drug concentrations that avoided significant cytotoxicity.

[0028] Example 2 Auranofin was administered at 0.625 μM to an AGS cell line (human gastric cancer cell line; Int J Cancer 99(5):644-651, 2002) persistently infected with EBV. This EBV-infected AGS cell line has traditionally been used as a model experimental system for the study of gastric cancer associated with latently infected EBV. Two days after administration, we measured the EBV genome copy number and the mRNA copy number of EBV-encoded viral genes per EBV genome to gain further insight into the inhibition of viral replication. Figure 4a shows the EBV genome copy number normalized to the GAPDH gene copy number in the host cells, confirming that auranofin administration significantly reduced the number of viral genome copies replicated in EBV-infected cells. Figure 4b shows the relative expression level of the BART gene per EBV genome. The relative expression level was determined based on the mRNA copy number. BART is a viral gene known to be expressed primarily during the latent phase of the EBV infection cycle and produces multiple microRNAs rather than encoding proteins. Auranofin administration significantly increased the relative expression of the BART gene. The expression of the early lytic phase genes BZLF1 and BRLF1 was also enhanced by auranofin administration (Fig. 4c). In contrast, the expression of BLLF1, a late gene expressed during the phase in which viral genome replication and virus particle production are particularly active, was suppressed by auranofin administration (Fig. 4d). These findings suggest that auranofin may inhibit EBV replication and, ultimately, the spread of infection, by suppressing late genes.

Claims

1. A composition for inhibiting Epstein-Barr virus replication, comprising fenofibrate.

2. 10. The composition of claim 1, which is a pharmaceutical composition for administration to a subject in need of inhibiting Epstein-Barr virus replication.

3. A composition which is a pharmaceutical for inhibiting Epstein-Barr virus replication, comprising auranofin, said composition being administered to a subject in need of inhibiting Epstein-Barr virus replication (excluding subjects with B-cell malignancies).

4. The composition of claim 2 or 3, wherein the subject has a primary Epstein-Barr virus infection.

5. The composition of claim 2 or 3, wherein the subject has a latent infection or reactivation of Epstein-Barr virus.

6. 4. The composition of claim 2 or 3, wherein the subject is an Epstein-Barr virus-infected patient having a disease selected from the group consisting of infectious mononucleosis, chronic active Epstein-Barr virus infection, lymphoproliferative disease, hemophagocytic syndrome, and autoimmune disease.

7. A pharmaceutical composition comprising fenofibrate for the treatment or prevention of a disease caused by Epstein-Barr virus infection, wherein the disease is infectious mononucleosis, chronic active Epstein-Barr virus infection, lymphoproliferative disease, hemophagocytic syndrome, or an autoimmune disease.

8. A pharmaceutical composition comprising auranofin for the treatment or prevention of a disease caused by Epstein-Barr virus infection (excluding B-cell malignancies), wherein the disease is infectious mononucleosis, chronic active Epstein-Barr virus infection, lymphoproliferative disease, hemophagocytic syndrome, or autoimmune disease.

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