Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7135—Compounds containing heavy metals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

• This disclosure relates to gammaherpesviruses, and in particular to Epstein-Barr virus (EBV) in humans.
• EBV Epstein-Barr virus
• EBV infects humans and is associated with a variety of diseases, but there are multiple stages in the infection process. These stages include “primary infection,” “latent infection” in which the virus remains dormant in the host after primary infection, expressing only a portion of the viral genes but not necessarily causing disease, and “reactivation,” which is triggered by factors such as a weakened immune system in the host.
• EBV infection is not uncommon, and in fact, most adults are infected with EBV. It is known that EBV is transmitted from person to person through saliva. Primary infection often occurs during infancy, and in most cases is asymptomatic, but sometimes (especially when primary infection occurs after adolescence) it can cause a disease called infectious mononucleosis. It is thought that there is an incubation period of about 4 to 6 weeks between the actual viral infection and the onset of the disease. The main symptoms of infectious mononucleosis are fever, redness of the throat, and swollen spleen, and about 70% of patients also develop hepatitis.
• EBV mainly infects lymphocytes, including B cells, and can progress to lymphoproliferative disorders and hemophagocytic syndrome, so caution is required.
• EBV Individuals who have been infected with EBV for the rest of their lives will carry EBV as an infected person. EBV that is latent in adults can be reactivated by factors such as immunosuppression, aging, and stress. When EBV is reactivated, the virus particles and the viral genome contained therein replicate actively again, which can cause host cell lysis. EBV reactivation has been associated with a variety of diseases, including neurological diseases such as multiple sclerosis and Alzheimer's disease, as well as autoimmune diseases such as rheumatism, SLE, and Graves' disease, and even aftereffects of COVID-19 infection. In addition, EBV-associated lymphoproliferative diseases caused by the administration of immunosuppressants during transplants, methotrexate treatment in rheumatism patients, and the administration of biological agents to patients with inflammatory bowel disease are also clinically problematic.
• diseases including neurological diseases such as multiple sclerosis and Alzheimer's disease, as well as autoimmune diseases such as rheumatism, SLE,
• EBV-specific treatment has been established, and there is a strong need for the development of new anti-EBV drugs.
• EBV is a species-specific virus that infects only humans. For this reason, even now, more than half a century after the discovery of EBV, it is difficult to experimentally analyze EBV itself in individual animals or animal cells, preventing the analysis of events immediately after initial infection and the promotion of evaluation of therapeutic drugs.
• MHV68 a mouse virus belonging to the same gamma herpes subfamily, has a mouse infection process that is highly similar to the process that occurs in human infection with EBV, and the MHV68 infection system is accepted worldwide as an EBV infection model based on knowledge accumulated over many years (Non-Patent Document 1).
• the objective of this disclosure is to provide a novel antiviral active agent capable of inhibiting EBV replication.
• the present inventors have found that known drugs called auranofin and fenofibrate can inhibit or suppress the replication of human EBV and its mouse model, MHV68.
• the embodiments of the present invention are based on this discovery.
• Auranofin and fenofibrate are known as anti-inflammatory drugs for rheumatoid arthritis patients and antihyperlipidemic drugs, respectively.
• auranofin and fenofibrate can inhibit the replication of the novel coronavirus SARS-CoV2, respectively (Rothan et al., Virology 547 (2020) 7-11; Ehrlich et al., eLife 2023;12:e79946).
• SARS-CoV2 novel coronavirus
• a composition for inhibiting Epstein-Barr virus replication comprising auranofin, fenofibrate, or a combination thereof.
• the composition described in [1] which is a pharmaceutical composition for administration to a subject in need of inhibiting Epstein-Barr virus replication.
• composition according to any 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.
• 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, a lymphoproliferative disorder, a hemophagocytic syndrome, or an autoimmune disease.
• the line graphs show cell viability in 3T12 cells (left) or MLE12 cells (right) cultured in the presence of auranofin.
• 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.
• Line graphs show cell viability in 3T12 cells (left) or MLE12 cells (right) cultured in the presence of fenofibrate.
• Bar graphs show viral load, i.e. number of viral copies detected per ml of culture supernatant, in cells cultured in the presence of the corresponding concentrations of fenofibrate.
• the line graphs show cell viability in 3T12 cells (left) or MLE12 cells (right) cultured in the presence of dipyridamole, a known EBV replication inhibitor.
• 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 dipyridamole.
• FIG. 4 shows EBV genome copy number (a), relative BART gene expression (b), relative BZLF1 and BRLF1 gene expression (c), and relative BLLF1 gene expression (d) in EBV-infected AGS cell lines treated with solvent control (white) or auranofin (black).
• 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 different medical uses, and basic knowledge has already been accumulated regarding their safety, dosage forms and delivery routes, pharmacokinetics, etc.
• An embodiment 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 exemplified disease, or a method of inhibiting EBV replication or treating or preventing an exemplified disease using auranofin, fenofibrate, or a combination thereof.
• auranofin, fenofibrate, or a combination thereof in the manufacture of a medicament for inhibiting EBV replication or treating or preventing an exemplified disease.
• EBV “replication” refers to EBV actively increasing the copy number of the viral genome and viral particles, as will be understood by those of skill 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.
• composition of the present embodiment may be delivered to cells or tissues, for example, in vitro or ex vivo.
• the composition of the present embodiment may 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.”
• the composition may be administered to a subject via a suitable delivery route known to those skilled in the art for auranofin and fenofibrate.
• a suitable delivery route known to those skilled in the art for auranofin and fenofibrate for example, oral or intravenous administration is preferred.
• Other routes of administration are also contemplated, including inhalation, topical administration, and the like.
• EBV replication occurs primarily in epithelial cells (particularly pharyngeal epithelial cells) and/or B cells, and a person skilled in the art may select a suitable route of administration based on his or her ordinary knowledge.
• 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 by it.
• the subject is a subject with primary EBV infection.
• the composition can be used to treat infectious mononucleosis, and embodiments in which the composition is used to prevent infectious mononucleosis are also contemplated.
• the subject is a subject with latent EBV infection or reactivation. There are many known EBV-associated disorders or diseases in which EBV reactivation is thought to contribute to the onset of the disease, and the composition can be used to treat or prevent these disorders or diseases.
• the composition of this embodiment can be administered in the setting of such immunosuppressant administration, i.e., in a subject to which an immunosuppressant is administered, to suppress EBV replication or to prevent an EBV-associated disorder or disease.
• the composition is administered to reduce EBV replication in a subject in which EBV replication has already begun, or to treat an EBV-associated disorder or disease.
• 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.
• a disease selected from the group consisting of infectious mononucleosis, chronic active EBV infection, lymphoproliferative disease, hemophagocytic syndrome, and autoimmune disease.
• 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 be a patient with a neurological disease such as multiple sclerosis or Alzheimer's disease, or a patient with sequelae of a COVID-19 infection.
• the subject may not be a rheumatoid arthritis patient.
• the subject may not be a patient with dyslipidemia, hyperlipidemia, hypertriglyceridemia, or hypercholesterolemia.
• the subject may not be a patient with SARS-CoV2 infection. Inhibiting EBV replication in a patient may result in treating or preventing a disease identified as being caused by EBV replication.
• a pharmaceutical composition includes 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 disease, hemophagocytic syndrome, or an autoimmune disease.
• composition of the present embodiment may contain a pharma- ceutically acceptable carrier or excipient in addition to the active ingredients auranofin and/or fenofibrate.
• manufacture of the pharmaceutical of the embodiment may include combining auranofin and/or fenofibrate with a pharma- ceutically acceptable carrier or excipient.
• compositions or pharmaceutical composition for inhibiting EBV replication of the present embodiment may be provided in the form of a liquid formulation.
• 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 a semi-solid formulation.
• Example 1 The present inventors have found that auranofin and fenofibrate each have activity to inhibit EBV replication.
• MHV68-infected system which is an established model of primary EBV infection, in comparison with dipyridamole, a known EBV replication inhibitor, and that these drugs can also exhibit viral replication inhibitory activity in the context of EBV reactivation.
• Mouse 3T12 or MLE12 cell lines were cultured in multi-well 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 contained in living cells into a formazan compound that absorbs at a specific wavelength.
• the cell lines were infected with 10 pfu of virus and simultaneously administered the above concentrations of auranofin, fenofibrate, or dipyridamole.
• the viral genome copy numbers in the culture supernatants 3 days after administration were measured by quantitative polymerase chain reaction (qPCR).
• 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.
• a horizontal line corresponding to 80% cell viability is shown for reference.
• 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).
• Figure 2 shows an experiment similar to that shown in Figure 1, except that in Figure 2, fenofibrate was administered instead of auranofin.
• Figure 3 shows an experiment similar to that shown in Figure 1, except that in Figure 3, dipyridamole was administered instead of auranofin. Dipyridamole is a drug whose EBV replication inhibitory activity has been shown in Non-Patent Document 2.
• auranofin and fenofibrate each had viral replication inhibitory activity that was at least comparable to or greater than that of dipyridamole. Furthermore, these viral replication inhibitory activities were achieved at drug concentrations that avoided significant cytotoxicity.
• AGS cell line human gastric cancer cell line; Int J Cancer 99(5):644-651, 2002
• This EBV-infected AGS cell line has been used as a model experimental system for the study of gastric cancer associated with latently infected EBV.
• Two days after administration we attempted to gain further insight into the inhibition of viral replication by measuring the copy number of EBV genome and the mRNA copy number of EBV-encoded viral genes per EBV genome.
• Figure 4a shows the copy number of EBV genome normalized to the GAPDH gene copy number of the host cell, confirming that administration of auranofin significantly reduced the number of viral genome copies replicated in EBV-infected cells.
• Figure 4b shows the relative expression level of 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 mainly in the latent phase of the EBV infection cycle, and produces multiple microRNAs rather than coding for proteins.
• Administration of auranofin significantly increased the relative expression of the BART gene.
• the expression of BZLF1 and BRLF1, which are early lytic phase genes, was also enhanced by administration of auranofin (Fig. 4c).
• the expression of BLLF1 a late gene expressed in the phase in which viral genome replication and viral particle production are particularly active, was suppressed by administration of auranofin (Fig. 4d). This suggests that auranofin may inhibit EBV replication and thus the spread of infection by suppressing late genes.

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• 2024
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