TW202033202A - Antiviral agent for treatment or prophylaxis of alzheimer's disease and use thereof - Google Patents

Abstract

The present invention provides a novel drug useful for fundamental treatment and prophylaxis of Alzheimer's disease (AD). As a pharmaceutical composition for treating or preventing AD, an agent comprises a compound having antiviral activity against HHV-6A, HHV-6B and/or HHV-7, particularly foscarnet, as an active ingredient is used. The agent not only stopsthe progression of AD but may also be used for fundamental treatment. In addition, by using the DNA or protein originated from HHV-6A, HHV-6B and/or HHV-7 existing in the cerebrospinal fluid (CSF), plasma, blood, or in the saliva of a subject, or using an antibody or a fragment thereof specific for HHV-6A, HHV-6B and/or HHV-7 as an AD biomarker, it is possible to diagnose the risk of developing AD or the progression of AD, or to confirm the therapeutic effect of an antiviral agent used in treating AD.

Description

Antiviral agent for treating or preventing Alzheimer's disease and its use

The present invention relates to therapeutic drugs for cranial nerve diseases, especially Alzheimer's disease and their uses.

(The status and pathology of Alzheimer's disease)

Alzheimer's disease ("AD") is the disease that accounts for the majority of dementia. In 2015, the number of patients worldwide was about 3 million. It is predicted that the number of patients will exceed 100 million by 2050.

AD is a staged symptom progression. In addition to the progression of memory impairment, there are instrumental activities of daily living (ADL) disorder or basic ADL disorder, which will be diagnosed as intermediate or moderate. Furthermore, AD patients develop olfactory disorders in the early stages. When it becomes the late stage, it is accompanied by a severe decrease in ADL, complicated by contact, dysphagia, pneumonia, falls, fractures, etc., and enters the terminal stage. In addition, various peripheral symptoms appear between the middle and later stages, such as restlessness, depression, insomnia, excitement, irritability, wandering, hallucinations, delusions, etc. In addition, the accumulation of Aβ in the brains of AD patients (the appearance of aging plaques) is a phenomenon that was confirmed 15 years ago (Reference 1), but its cause is unknown so far.

Reference 1: RJ Bateman et al., N. Engl. J. Med. 367 (9): 795-804, 2012

(Causes of Alzheimer's disease)

In the brains of AD patients, the increase in amyloid β protein ("Aβ") and the accumulation of aggregates ("Aβ oligomers") are significant, showing that Aβ (especially containing 42 amino acids) The high soluble Aβ ₄₂ ) is the main component of the increase in aging plaques, and cerebral amyloid angiopathy. Through the increase and accumulation of Aβ/Aβ oligomers, the shedding of synapses, activation of microglia, inflammatory cytokines such as IL-6, IL-12, and TNF-α, or chemotactic mediators such as CCL2 It is believed that Aβ is an important factor in the onset of AD (Non-Patent Document 1, References 2 and 3).

Most patients with AD have Aβ attached to the cerebral blood vessels, causing inflammation and amyloid angiopathy (Reference 3). Amyloid angiopathy in the brain causes the fragility and vulnerabilities of the BBB (Reference 4), and promotes the migration of T cells or NK cells in the peripheral blood to the inflammation site.

Furthermore, AD showed hyperphosphorylation and agglutination of the Tau protein, which is the main component of microtubules, and the accompanying neurofibrillary changes, progression of nerve cell death, and brain (especially hippocampal gyrus) in patients with advanced symptoms. ) Atrophy or a large number of nerve cell deaths, etc. (Reference 5).

In addition, the excessive AD, which can be described as the cause of cell death, is the pathological "apoptosis" caused by the accumulation of Aβ (especially oligomers mainly composed of Aβ42) in the cells of the salt line (References 1, 5) . It has been reported that a large number of DNA fragments are observed in the brain of AD patients (Reference 5) as evidence that the cell death is shown. Furthermore, the regeneration of nerve cells in the hippocampal gyrus is drastically reduced (Non-Patent Literature 2).

Although the massive death and insufficiency of regeneration of nerve cells can be said to be the root cause of the pathology of AD, the relationship between the massive death of nerve cells and the insufficiency of regeneration is not yet fully understood.

Reference 2: S. West et al., Br. J. Clin. Pharmacol. 80 (2) 221-234, 2015

Reference 3: B Mroczko et al., International Journal of Molecular Sciences (19): 1-29, 2018

Reference 4: KA Jellinger et al., Journal of Alzheimer’s Disease 3 (2001):31-40, 2001

Reference 5: Masaki Ueno, "Clinical Neurology" Volume 57 No. 3, 95-109, 2017

(Development target of AD drugs)

From the viewpoint that it is related to the increase or accumulation of Aβ (including its oligomers) in the ventricle of AD, or the modification of Tau protein, the study of Aβ or Tau protein as the target AD therapeutic drug, Development is proceeding on a worldwide scale (Non-Patent Document 3, Reference Document 6). Specifically, Aβ formation/deposition, Aβ-producing enzyme β secretase (BASE-1) and γ secretase, Aβ oligomer formation, phosphorylation of Tau protein, etc. are used as targets, some of which are used in AD The effect is shown in the experiment of disease model animals.

However, most of the new AD drugs that target Aβ have not been successfully developed due to effectiveness or safety issues, and have been developed based on new perspectives, such as disease-modifying therapies (DMTs, Disease-Modifying Therapies) or according to the latest Discover the development of AD drugs (iPS cells etc.) (Non-Patent Document 3). Among them, although it falls into the category of DMTs, there are also innovative drugs that regard AD as a chronic inflammatory disease, and target factors that cause inflammation in nerve tissue (Reference 6). However, as a DMT drug for AD, commercialization has not yet been completed.

Reference 6: AA Fabregat et al., CNS Drugs (2017) 31: 1057-1082, 2017

The onset or worsening of AD is related to chronic inflammation that occurs in the brain, and it has gradually become a common theory. DAMPs (Damage or Danger-Associated Molecular Patterns) are referred to as the factors that cause it ( Non-Patent Documents 4 and 5).

DAMPs are a group of substances released by cells that have been injured and died for any reason. Their sources are nucleus, cytoplasm, mitochondria, endoplasmic reticulum, etc., and the released substances are also diverse (Non-Patent Document 5, Reference 7). Among them, as persons related to AD, Aβ, calcium (reference 8), HMGB-1 (reference 9), TDP-43 (reference 10), GSK-3β (reference 11), bran Amino acid/glutamine (reference 12), ATP (reference 13), bacteria/virus (references 14, 15), etc. Furthermore, infections caused by pathogens such as viruses are also called PAMPs (Reference 7).

The majority of DAMPs are not caused by "apoptosis" due to cell death arranged by the established process, but when the "necrosis" caused by any disease as the main factor is released outside the cell. Furthermore, it is known that virus-infected cells express chemokines (CCL2/MCP-1, etc.) or cytokines (IFN-γ, IL-6, TNFα, etc.) also trigger DAMPs (such as HMGB-1, etc.) ( Non-Patent Document 6).

Reference 7: Keji Yoshida, "Surgery and Metabolism/Nutrition" Volume 48, Number 6: 247-249, 2014

Reference 8: Y Wang et al., J. of Alzheimer’s Disease & Parkinsonism, 7 (5): 1-15, 2017

Reference 9: K Fujita et al., Scientific Reports 6 (31895): 1-15, 2016

Reference 10: XL Chang et al., Molecular Neurobiology 53 (5): 3349-3359, 2016

Reference 11: ML Martin et al., frontiers in Molecular Neuroscience 7 (46): 1-11, 2014

Reference 12: C Madeira et al., frontiers in Psychiatry 9 (561): 1-9, 2018

Reference 13: Sato Hidetoshi and others, "Japanese Pharmacology" 136: 93-97, 2010

Reference 14: M Sochocka et al., Current Neuropharmacology, 15 (7): 996-1009, 2017

Reference 15: Haruka Minming, "Surgery and Metabolism/Nutrition" 51(1): 1-7, 2017

(AD and nerve cell death)

In the past, when it was considered as an adult, nerve cells would not regenerate. However, it has been gradually understood that there are neural stem cells in the dentate gyrus of the hippocampus or the hypooptic area of the back of the brain, and nerve cells can be regenerated throughout their lives (Non-Patent Document 7). This fact shows the importance of inhibiting the death of neural stem cells or cell death in the process of differentiation by seeking to stop the maturation and prolong the life of nerve cells that are about to undergo apoptosis in the treatment of AD.

Furthermore, although it is known that neural stem cells present in the lower part of the posterior ventricle differentiate into nerve cells and migrate to the olfactory bulb, it is unclear whether they become other brain tissues, such as nerve cells in the cerebral neocortex.

(Viral infection as a cause of dementia)

It has been reported that the onset or worsening of dementia is related to viruses or fungi (Ref. 13), especially because of the severe blisters caused by reactivation of herpes simplex virus type 1 (HSV-1) in the brain The clinical images of herpes virus encephalitis (HSVE) are close to AD and other reasons. HSV-1 is a strong candidate for dementia, especially the cause of AD (Non-Patent Documents 8, 9, Reference 17). Furthermore, There is also a study in which patients who were treated with antiviral agents during HSV-1 infection had fewer dementias than untreated patients (Ref. 18).

However, HSV-1 is a risk factor for the onset of AD. Although it was alleged that HSV-1 infection caused the accumulation of Aβ or hyperphosphorylation of Tau protein decades ago (Non-Patent Document 8), HSV- 1 is the proof of the main cause of AD.

Reference 17: SA Harris et al., Frontiers in Aging Neuroscience, 10 (48): 1-24, 2018

Reference 18: Tzeng NS. et al., Neurotherapeutics, 15 (2): 417-429, 2018

Other than HSVE, clinical images similar to AD show that the reactivation of latent virus is caused by "HHV-6 encephalitis". The main cause of the disease is human herpesvirus type 6 B (HHV-6B) (Reference 19) .

HHV-6 encephalitis is caused by the latent infection of HHV-6 (especially HHV-6B) in the brain when the immunity of hematopoietic stem cells or bone marrow or organs is reduced during the initial infection in infants and young children. Reactivation occurs when hyperactivity (massive proliferation of the virus) occurs (references 20 to 22). However, there are few cases of encephalitis in healthy people. This shows that HHV-6 (A and B) in the brain tissue of healthy people limits the proliferation of the virus even if it is reactivated, and does not cause detectable clinical symptoms that are suspicious of health.

In addition, whether the brain cancer that occurs after bone marrow or organ transplantation is caused by HHV-6 (A and B), if the existence of other causes of viruses can be denied, the PCR method can be used to check the HHV in the cerebrospinal fluid -6 (A and B) the presence and number of specific NA to be determined (Reference 24).

Reference 19: Kawamura Yoshiki and others, "Japanese Clinics" 69 Vol. 3, 423-427, 2011

Reference 20: A Ansari et al., Emerging Infection Disease 10 (8), 2004

Reference 21: Masao Ogata, "Clinical Blood" Volume 57, No. 3: 298-306, 2016

Reference 22: LD Bolle et al., Clinical Microbiology Reviews, Jan: 217-245, 2005

Reference 23: J Ongradi et al., J. Neurovirol. 23: 1-19, 2016

Reference 24: Kyouko Taya and others, National Institute of Infectious Diseases Pathogen Inspection (Detection) Manual "Sudden Eruption", http://www.niid.go.jp/niid/ja/labo-manual. html

(Human herpes virus type 6 (HHV-6) and type 7 (HHV-7))

HHV-6 was found in 1986, and HHV-7 was a DNA virus with double-stranded DNA as its genome discovered in 1990. It belongs to subspecies B of the Herpesvirus family.

HHV-6 has two types of HHV-6A and HHV-6B (now it is believed that it is not a subspecies but a separate virus). The two types have about 90% identity in base sequence and are duplicated with human chromosomal telomeres The sequence ([TAAGGG]n) is similar to the repeat sequence ([TAACCC]n) at both ends of the genome (References 25, 26).

Almost all people are infected with HHV-6 (A and B) or HHV-7 for the first time in infants and young children. As the latent infection of HHV-6A and HHV-6B, CNS tissues (brain glial cells or hippocampal gyrus, nerve Cells, etc.) (Reference 27). Furthermore, HHV-7 may be latent or infect the salivary glands or olfactory bulb.

As the main recipients of host cells invaded by the above viruses, it is known that HHV-6A is CD46 (expressed in almost all cells), and HHV-6B has CD134 in addition to CD46 (expressed in CD4-positive and/or CD8-positive T cells). ), HHV-7 is CD4 or CD8 (shown in activated T cells, etc.) (References 25, 26).

After the initial infection, HHV-6 (A and B), which cannot be ruled out by the immune mechanism, lurks in the host cell. The method uniquely integrates the viral genome with the telomeres of the human chromosome and lurks. Furthermore, only one HHV-6 (A or B) gene body is incorporated into a chromosome of the host cell, and the chromosomes incorporated as a viral gene body are known as X, 1, 6, 7, 9, 10, 11, 12, 17 , 18, 19, and 22 chromosomes (reference 28), it is impossible to confirm the latency in other chromosomes.

About 1% of all human beings inherit the gene body of HHV-6A or HHV-6B by inheritance, but the pathological effects of this virus (iciHHV-6A and iciHHV-6B) are almost unknown at present. However, it is referred to as an important factor of activation.

Reference 25: H Agut et al., Clinical Microbiology Reviews 28 (2): 313-335, 2015

Reference 26: Tang Huamin and others, "Virus" 60 (2): 221-236, 2010

Reference 27: T Yoshikawa et al., Arch. Dis. Child. 83: 170-171, 2000

Reference 28: DA Clark, Clin. Microbiol. Infec. Vol. 22: 333-339, 2016

HHV-7 and HHV-6 (A and B) have the same repeated base sequence ([TAACCC]n) located at both ends of the gene body, which is related to the onset of diseases similar to HHV-6 encephalitis (Reference 29 ), but it is not yet clear whether it is integrated with the telomeres of human chromosomes as in HHV-6 (A and B).

As a disease caused by HHV-7 infection, it is known that HHV-7 in infants and young children rarely causes sudden rash or chronic fatigue syndrome after initial infection (Ref. 26). However, like HHV-6 (A and B), the virus has no harmful effects on the human body during latent infection. Unless it proliferates rapidly through reactivation, it is believed that most people with latent infection of the virus will have no problems throughout their lives.

Reference 29: M Parra et al., Virology Journal, 14 (97): 1-5, 2017

HHV-6B is not only HHV-6 encephalitis, but also a major virus that causes idiopathic rash in infants or encephalitis at the time of initial infection (References 25, 26). Furthermore, almost all of the HHV-6 that causes the onset of clinically observed pathological effects are HHV-6B, and the detailed reasons are still unclear.

The pathology of HHV-6A or HHV-7 upon reactivation is still unclear, but HHV-6A and HHV-6B infect cells and cause apoptosis (Non-Patent Document 10, Reference 30), and HHV-6A may Co-infection with HHV-7 (Ref. 31). On the other hand, co-infection with HHV-6A and HHV-7 has a mechanism to escape from NK cells (Non-Patent Document 11).

Reference 30: B Gu et al., Virology Journal 8 (5) 5: 1-10, 2011

Reference 31: M Ihara et al., Microbiol. Immunol. 45 (3): 225-232, 2001

(HHV-6 Encephalitis and AD)

A typical case of the initial symptoms of HHV-6 encephalitis, which can answer events that occurred in the distant past, but cannot remember short-term memory (short-term memory impairment) such as daily activities, and is not clear about the place of one's own room or toilet. Such disorientation Disorder (dementia), similar to the typical symptoms of early AD.

Furthermore, as the frequency of initial symptoms, in domestic surveys, it was reported as consciousness disturbance (62%), disorientation (52%), memory impairment (48%), cramps (32%), sensory disturbance (22%) ), autonomic nerve disorder (6.2%) (Ref. 32). The prevalence period is reported to be 12 to 30 days after organ transplantation (Ref. 33), and the possibility of serious sequelae or death is also high when the start of treatment is delayed.

In the head MRI examination of patients with HHV-6 encephalitis, bilateral abnormalities in the limbic region of the brain (the hippocampal gyrus and amygdala on the inner side of the lateral head) are seen (Ref. 34). This is seen in typical pathological observations in AD. However, even though HHV-6 (A and B) or HHV-7 infection is a risk factor for the onset or exacerbation of AD, there is no investigation or research report that refers to the direct cause of AD.

Furthermore, in June 2018, it was disclosed that the number of DNA of HHV-6A and HHV-7 in the brains of AD patients (parahippocampal gyrus, etc.) is approximately twice that of non-patients (Non-Patent Document 12) , But the relationship between AD and the DNA concentration/number of these viruses is not yet clear.

Reference 32: Japanese Society for Hematopoietic Transplantation "Guidelines for Hematopoietic Cell Transplantation, Prevention and Treatment of Viral Infection HHV-6", 2018, http://www.jshct.com/uploads/files/guideline/01_03_03_hhv6/pdf

Reference 33: DM Zerr et al., Clinical Infectious Diseases. 2002; 34: 309-3017, 2002

Reference 34: T Noguchi et al., Am. J. Neuroradiol. 27: 2191-2195, 2006

These results have lasted more than 30 years, and AD therapeutic drugs based on various hypotheses have been researched and developed in research institutions such as pharmaceutical companies or universities (Non-Patent Documents 3, 4, References 6, 35), but there is currently no known development of any AD therapeutics targeting HHV-6 (HHV-6A and/or HHV-6B) or HHV-7. Furthermore, no attempt has been made to target the virus in AD patients The treatment trial.

Furthermore, the compound or its pharmaceutical composition (preparation) that has antiviral effect on HHV-6 (A and B) or HHV-7, for example, includes foscarnet, famciclovir, acyclovir (aciclovir) ), valaciclovir (valaciclovir), ganciclovir (ganciclovir), bisphosphonate derivatives of cidofovir, anti-CD40 antibody, pyridoquinoxaline, pyrazolopyridine derivatives, fluor Previous patents for antiviral agents such as luciferin compounds, SITH-1 inhibitors, nanometers, emulsified compositions, gremlin derivatives, etc. did not find that AD is an adaptive disease patient.

Reference 35: Y Dong et al., International J. Molecular Sciences 20 (588): 1-24, 2019

HHV-6 encephalitis can be indicated as medicines (including anti-herpes virus agents). Although it has not been seen in the world, it has been used in clinical sites for more than ten years. Foscarnet and ganciclovir have been used as The main preparation is still used for treatment outside of indications (Non-Patent Document 13, References 20 and 32).

Based on these results, in February 2018, the "Guidelines for Hematopoietic Cell Transplantation" (Ref. 32) formulated by the Japanese Society for Hematopoietic Transplantation formulated "basic treatment for HHV-6 encephalitis. The full dose (60mg/kg, every 8 hours, 3 times a day) is administered, and the treatment period is at least 3 weeks."

[Prior Technical Literature]

[Non-Patent Literature]

[Non-Patent Document 1] Takahiko Tokuda, "Kyofu Medical History", Volume 125, No. 12: 797-806, 2016

[Non-Patent Document 2] EP Moreno-Jimenez et el., Natura Medicine (25): 554-560, 2019

[Non-Patent Document 3] J Cummings et al., Alzheimer’s & Dementia: Translational Research & Clinical Interventions 4 (2018): 195-214, 2018

[Non-Patent Document 4] C Venegas et al., Journal of Leukocyte Biology 101: 1-12, 2017

[Non-Patent Document 5] E Venereau et al., frontiers in Immunology. 6 (422): 1-11.2016

[Non-Patent Document 6] M Magna et al., Molecular Medicine 20: 138-146, 2014

[Non-Patent Document 7] KL Spalding et al., Cell 153 (6): 1219-1227, 2013,

[Non-Patent Document 8] MA Wozniak et al., PLPS ONE vol. 6(10): 1-15, 2011

[Non-Patent Document 9] RF Itzhaki, FASEB Journal, vol.31, 3216-3226, 2017

[Non-Patent Document 10] Y Inoue et al., Journal of Virology May, 1997: 3751-3759,1997

[Non-Patent Document 11] P Secchiero et al., Blood 98 8): 2474-2481, 2001

[Non-Patent Document 12] B. Readhead et al., Neuron 99 (1), 64-82, e7, 2018

[Non-Patent Document 13] M Ogata et al., Biol. Blood Marrow Transplant. 24: 1264-1273, 2018

[Non-Patent Document 14] F Raffi et al., Antimicrobial Agents and Chemotherapy, 37 (9):1777-1780, 1993

[Non-Patent Document 15] J Sjovall et al., Clinical Pharmacol. Therapy, 44: 65-73, 1988

[Non-Patent Document 16] M Yoshida et al., Antiviral Research 40 (1998): 73-84, 1998

[Non-Patent Document 17] ED Clercq et al., Rev. Med. Virol. 2001;11: 381-395, 2001

[Non-Patent Document 18] A Kaczmarek et al., Immunity 38, Feb: 209-223, 2013

Although various hypotheses have been proposed as the cause of Alzheimer's disease ("AD"), the development of AD therapeutics based on this hypothesis has been carried out in Europe, America and Japan for decades. However, even for the most anticipated new drug development with Aβ as the target, there are no practical compounds yet. The development of radical AD therapeutics that prevent AD, stop progression, and achieve improvement are urgently desired.

The present inventors focused on the related points of HSV-1 infection of the latent virus in the onset of dementia, and considered the treatment of AD by targeting the latent virus in the brain that does not usually infect symptoms.

That is, although latently infecting glial cells or nerve cells in the brain, the target is the latent virus that is accompanied by infection but does not show inflammatory symptoms (fever, redness, etc.) under these conditions, and inhibits it through antiviral agents. Prevention and treatment of reactivated AD. Antiviral agents are used in the treatment and prevention of AD because the reactivation of the latent virus in the brain promotes the production of Aβ as a natural immune response, and furthermore, the pathology (inflammation or cell death, etc.) caused by the infection of the activated virus ) Is deeply related to the onset and worsening of AD.

Latent infection with a virus or its reactivation is a report established by the joint action of multiple viruses (Non-Patent Document 15). The inventors focused on this point and considered that the brains of AD patients, especially the hippocampus, are known as HHV-6A, HHV-6B, and HHV-7 (hereinafter referred to as "HHV- 6/7”) and the infection symptoms at the time of reactivation are highly likely to worsen or become obvious.

The production of Aβ is assumed to be a natural immune response to viruses that appear in the brain. When HHV-6/7 is reactivated, Aβ produced and released by nerve cells attaches to the surface of these viruses and prevents movement. It is helpful for the phagocytosis of the virus through the microglial cells that act as macrophages in the brain. Furthermore, viruses that evade microglial cell phagocytosis may be absorbed and accumulated in nerve cells alone or in a complex with Aβ through endocytosis.

If the above hypothesis is correct, it can explain the mechanism of Aβ accumulation in the nerve cells of AD.

In other words, the accumulation of Aβ in the cells of AD patients is at least related to the infection associated with the reactivation of HHV-6/7 latent infection in the brain. If the reactivation and infection are inhibited, it is possible to prevent or treat the Aβ-related AD development. Disease (Figure 1).

When the Aβ-producing enzyme (β-secretase, etc.) is inhibited and only the production of Aβ is suppressed, the defense against the virus is reduced, and there is a possibility that the reactivated virus may be allowed to proliferate.

The inventors considered the reactivation of latent viruses in the brain, especially HHV-6/7, if it is the root cause of Aβ production, the elimination of related latent viruses (specifically, infection during reactivation and inhibition of virus proliferation) ), inhibit the production of Aβ and its oligomerization, the accumulation of Aβ (oligomers) in nerve cells, and the rapid increase in the concentration of Ca ⁺⁺ caused by it, and various important factors that cause nerve cell death, including Possibility to become a fundamental treatment for AD (Figure 2).

Here, in order to verify the above hypothesis, imitating HHV-6 (A and B) and/or HHV-7 (hereinafter, also referred to as "HHV-6/7") in the central nervous system of human beings are potentially infected As a model cell, the following experimental system was constructed using cultured human nerve cell lines infected with HHV-6/7. Moreover, the animal model test system has not been constructed. This is because HHV-6/7 is a human-specific herpes virus, and a suitable animal model cannot be obtained.

1) Preparation of virus solutions of HHV-6A, HHV-6B and HHV-7

2) Modulation of Aβ oligomers

As the Aβ used in the test, the main constituent molecule of Aβ oligomer, which is the main component of aging plaque, was selected as soluble Aβ ₄₂ composed of 42 amino acids.

3) Test cells

As a human-derived nerve cell line, select the standard SH-SY5Y strain (neuroblast cell line derived from the class)

4) Test sample group

In the experiment, the following sample groups were constructed. Regarding the Aβ supplement group at 6 concentrations (0, 0.1, 1.0, 10, 100, 1000 nM), the test cells were cultured for 24 hours and 48 hours. Moreover, each virus is a virus solution adjusted by the process of Example 1. Furthermore, the intravenous injection preparation "Foscavir (registered trademark)" with the same ingredient as foscarnet as the only active ingredient.

5) Summary of test methods

Add HHV-6/7 virus solutions and multiple concentrations of Aβ oligomers to the medium in the test cell culture. The HHV-6/7 infected cell line takes the Aβ of the "virus complex" into the cells, and studies Does Aβ accumulate excessively in the cell? Furthermore, it was examined whether Aβ outside the cell and Aβ taken into the cell have cytotoxicity (inhibition of proliferation, cell death, etc.).

The results of the test showed that, regardless of the presence of virus (HHV-6/7) outside the cell, Aβ _{42 in the} culture medium (extracellular) was taken up into the test cell. However, when the concentration of Aβ is 0.1 to 10 nM, the intracellular concentration of Aβ ₄₂ is in the mixed solution of HHV-6A and HHV-7, the mixed solution of HHV-6B and HHV-7, and the virus solution of HHV-7 alone. Although it showed a decreasing tendency, no such decreasing tendency was observed in the virus solution-unadded group (Figures 7 and 8). This means that the extracellular virus system binds to Aβ ₄₂ and inhibits the migration of Aβ _{42 into} the cell.

In contrast, the addition of the HHV-6/7 virus solution under the test conditions did not show an increase in intracellular Aβ.

Furthermore, in an experiment to study the number of virus (HHV-6) DNA in infected test cells, although the number of DNA decreased in proportion to the concentration of Aβ ₄₂ added, it was observed in the non-added Aβ ₄₂ population. To this reduction (Figure 8). This means that the extracellular Aβ system inhibits the uptake of viruses existing outside the cell into the cell.

The above test results show that Aβ ₄₂ has a defensive effect on virus infection (toward the inhibition of virus uptake in cells) and also has a defensive effect on HHV-6/7. However, the "virus Aβ complex" (the virus and the bound Aβ ₄₂ ) is taken into the cell, and the accumulation of Aβ ₄₂ in the cell can not be verified under experimental conditions.

So far, cells infected with HHV-6/7 cause apoptosis (R Ichimi et al., Journal of Medical Virology, 58(1): 63-68, 1999, and non-patent documents 10 and 11, reference 30 ), the presence or absence of death of infected cells was verified through each virus related to the above test (Example 5).

Regarding HHV-6A, the uninfected HSB-2 strain (human T lymphocyte cell line), HHV-6B and HHV-7-related uninfected Sup-T1 strain (human T lymphocyte-derived T cell line) In (Example 1), each obtained virus solution was added to observe the presence or absence of virus infection and the morphological changes of the infected cells (Example 5).

As a result, in the uninfected cell population of HHV-6A (Fig. 10-1), the shape or size of each cell was uniform, and almost no dead cells were observed. However, in the infected cell population (Figure 10-2), hypertrophic cells and dead cells were scattered.

In addition, regarding HHV-6B and HHV-7, in the uninfected Sup-T1 strain population (Figure 11-1), no dead cells were seen, and the cell shape and size were uniform. However, in the HHV-6B-infected cell population (Figure 11-2) and HHV-7-infected cell population (Figure 12), except for a few dead cells, clumped cells and hypertrophic cells were scattered on the cell surface.

The inventors of the present invention found from the above test results that T cells infected with HHV-6/7 also cause necrosis other than apoptosis. These two different types of cell death, especially the pathology that causes necrosis, are different from the reactivation of the above viruses. The pathology of Aβ (selective neurological disease, cell death, obstruction of Ca ⁺⁺ constancy, synaptic damage, activation of microglia or promotion of astrocyte response, increase in insulin resistance, etc.: Reference 3) synergy is the fundamental cause of AD.

That is, in response to the mild inflammation (HHV-6 encephalitis) associated with the reactivation of HHV-6/7 produced by the hippocampal gyrus with increasing age, in addition to the production of Aβ from nerve cells, T cells are activated Migrate through the weakened BBB to reach the site. At this time, part of the T cells infected by the virus are necrotic, and the HMGB-1 type of DAMP released by the dead cells or the infected cells (not limited to T cells) exhibit the effect of the CCL2 type of chemotactic interleukin, resulting in cell disorders The migration/agglutination of immune-related cells such as sex T cells and NK cells toward the site of infection is enhanced. As a result, in AD patients, the local cell death of the site infected with the reactivated virus (especially the hippocampal gyrus) continues, and when nerve cell death exceeds the regenerative capacity, the nerve function is irreversibly damaged ("HHV -6 encephalitis cascade hypothesis") is established.

Based on the above, it was discovered that by using various herpes virus agents that have excellent effects on HHV-6 (A and B) and HHV-7, not only the progression of AD can be stopped, but fundamental treatment can be achieved in a short period of time (3 to 6 months). And completed the present invention.

In the present invention, it is believed that AD develops symptoms and worsens through the following process ("HHV-6 encephalitis cascade hypothesis").

1) Almost all people are infected with HHV-6 (A and B) and HHV-7 when they are young. The virus, especially HHV-6 (A and B), is latent in CNS tissues (glia cells of the brain, especially Astrocytes and hippocampal gyrus or olfactory bulb with regenerable nerve cells, etc.) (JM Reynaud et al., ISRN Virology 2013, vol. 2013: 1-11, 2013).

2) In human cells, the above-mentioned virus not only infects/latents in immune cells such as monocytes or CD4 or CD8-positive T cells, but can also proliferate in these cells (Ref. 34). Therefore, by reactivating the CCL2 and other chemokines produced by the infected cells, the main receptors of the virus are CD46 (HHV-6A), CD134 (HHV-6B), and CD4 (HHV-7). When activated T cells and the like migrate to the infection site, the virus will proliferate instead of phagocytosis.

3) In the hippocampal gyrus, the neural stem cells of the dentate gyrus differentiate toward precursor nerve cells, granular cells, and pyramidal cells to maintain the constant number of nerve cells.

4) Since the latent DNA virus is reactivated during the differentiation or division of the host cell, the HHV-6 of the DNA virus latent in the neural stem cells or precursor nerve cells of the hippocampal gyrus is highly likely to be reactivated.

5) Latent HHV-6 is reactivated, causing mild inflammation (HHV-6 encephalitis) accompanied by infection nearby. Aβ is produced/secreted as a natural immune response to the nerve cell line that is aware of the virus infection. It captures the virus or senses it by astrocytes and transmits it to the microglia cells that act as macrophages in the brain. Let this process be done. At this time, due to the expression of inflammatory cytokines such as TNF-α or IFN, or chemotactic mediators such as CCL2, T cells are activated and migrate to the site of infection through the BBB.

6) Due to the latent method of HHV-6, the number of viruses that can be latent in a cell is small. Therefore, the number of reactivated viruses is also small. Usually, microglial cells or T cells that act as macrophages in the brain are phagocytosed. As a result, viral infections accompanying HHV-6 reactivation do not cause clinical symptoms of the disease (fever, etc.) except in exceptional cases.

This means that viral encephalitis caused by HHV-6/7 rarely develops. However, Aβ plaques (aging plaques) containing the viral DNA as residues of virus reactivation can be scattered in the infection site in the brain. In AD patients, the accumulation of Aβ plaques (aging plaques) in the brain began more than 15 years before the onset of the disease, which is possible from the above explanation.

7) With aging, the first vulnerable BBB is the hippocampus BBB (A Montagne et al., Neuron, 85(2): 296-302, 2015). Therefore, when inflammation is enhanced by DAMPs such as HMGB-1 in the hippocampus, NK cells or activated T cell lines in the peripheral blood migrate to the brain through the weakened BBB by the inducement of chemokines such as CCL2. Inflammation site. In particular, since T cells can easily pass through the BBB (Masui Matsui, Clinical Neuro 53(11): 898-900, 2013), the migration of activated T cells toward the site of infection is hyperactive.

8) As a result, the number of HHV-6 (A and B) that can proliferate in activated T cells expressing CD4 or CD8 increases, and the infection is expanded/severe. Furthermore, as a result of the migration of NK cells, which mainly attack the role of pathogens such as viruses in the living body, cell death (mostly apoptosis) via NK cells or barrier T cells (expressing CD8) is enhanced.

9) In other words, reactivation of inflammation caused by infection of HHV-6 (A and/or B) → migration of activated T cells → infection of T cells → necrosis of infected T cells → release of DAMPs → enhancement of inflammation/infected cells (Including nerve cells) Apoptosis/necrosis→release of DAMPs→enhancement of migration/infiltration of activated T cells/NK cells toward the site of infection→further cell death, the negative cycle of the above-mentioned methods, makes nerve function at the site of infection The death of cells (including astrocytes and nerve cells) is increased.

In particular, when the olfactory bulb and other HHV-7 that are latently infected are reactivated and moved to the hippocampal gyrus, or reactivated in the hippocampal gyrus, through co-infection with HHV-6A, the infected cells can be used by the surrounding uninfected nerve cells. TRAIL manifests and expands cell death via NK cells. As a result, in response to viral infection and the effect of Aβ in tissues with neural stem cells or precursor cells (hippocampal gyrus and limbic system), encephalitis (HHV-6) accompanied by reactivation of HHV-6 (A and B) Encephalitis (encephalitis) and other pathologies become obvious, the cell death exceeding the regenerative capacity progresses slowly and surely, and the nerve function is gradually damaged and AD develops.

The outline of the process of reaching the onset of AD in the "HHV-6 Encephalitis Cascade Hypothesis" is as follows (Figure 1).

From the above, it is understood that the present invention includes the following inventions.

[1] A pharmaceutical composition for the treatment or prevention of Alzheimer’s disease ("AD") against human herpesvirus type 6 A and B (hereinafter collectively referred to as "HHV-6") and/or type 7 ( "HHV-7") A pharmaceutical composition containing a compound with antiviral activity as an effective ingredient and a pharmaceutically acceptable carrier and its use.

For the treatment (including prevention) of Alzheimer's disease ("AD"), an effective amount of a compound having antiviral activity against "HHV-6" and/or "HHV-7" is expected to be further used in an equivalent effective amount It is a compound with a variety of antiviral effects that is active against herpes simplex virus type 1 ("HSV-1") and cytomegalovirus ("CMV") that cause encephalitis and other central nervous disorders.

[2] The aforementioned compound is the pharmaceutical composition described in the aforementioned [1], which is a compound having antiviral activity against HSV-1 and CMV, and its use.

Herein, the pharmaceutical composition of the present invention is active not only against "HHV-6" and "HHV-7", but also against HSV-1 and CMV, which cause central nervous system diseases such as encephalitis, in equivalent or close to effective amounts. , That is, compounds with multiple antiviral effects as effective ingredients.

[3] The aforementioned compound is a compound that binds to the pyrophosphate binding site of a DNA polymerase derived from a target virus and selectively inhibits the proliferation of the virus, the pharmaceutical composition described in [1] or [2] and its use.

[4] The pharmaceutical composition described in any one of the foregoing [1] to [3], and the use thereof, which is a pyrophosphoric acid analog of a derivative of phosphinyl acetic acid or phosphinyl carboxylic acid or the like.

In addition, it is known that phosphonoacetic acid or its derivatives have antiviral activity similar to phosphonocarboxylic acid or its derivatives.

[5] The aforementioned compound is the aforementioned phosphinyl carboxylic acid or its salt, or solvate thereof (these are hereinafter referred to as "phosphinyl carboxylic acid derivatives") represented by the following basic structural formula (Formula 1) [ 1] The pharmaceutical composition described in any one of [4] and its use.

In addition, the best compound among the "phosphinyl carboxylic acid derivatives" of (Formula 1) is the hexahydrate of 3 sodium phosphinyl carboxylic acid represented by (Formula 2) (hereinafter referred to as "phosphonic acid"), (JNN )/INN is marked as Fosearnet Sodium.

[6] The aforementioned pharmaceutical composition contains an injection containing foscarnet as an active ingredient, and an effective amount of foscarnet is exemplified by 60-180 mg/kg/day divided into 2 or 3 times a day, and administered via intramedullary cavity Or intravenous infusion of the medical composition described in [5] above and its use, which is continuously administered for 3 to 10 days.

Here, when the injection of the present invention using foscarnet as an active ingredient is used as a pre-emptive therapy for AD, each of HHV-6A, HHV-6B, and HHV-7 in the cerebrospinal fluid and/or blood The amount of viral DNA is reduced to below the level of healthy people, and it is expected to be below the detection level. For example, the effective amount of foscarnet is 60-180 mg/kg/day, divided into 1 or 3 times a day, and administered via intramedullary cavity It is administered by intravenous or intravenous injection for a period of 3 to 10 days, and it is expected that the administration will continue for 3 to 5 days.

Moreover, in order to check the reduction of the amount of viral DNA and its maintenance status, the recovery status of the patient’s cognitive function, and to prevent or regulate the maintenance of foscarnet tolerance or the occurrence of serious side effects such as renal impairment, the first-in-class therapy can be appropriately adjusted The period of time and the dosage of foscarnet per time or each day.

[7] The aforementioned pharmaceutical composition is an injection containing foscarnet as an effective ingredient for the purpose of preventing the reactivation of each virus. The effective amount of foscarnet is 60-120 mg/kg/day, via intravenous injection, intramuscular injection or Subcutaneous injection, continuous administration for 2 to 6 months, the medicinal composition for maintenance therapy after the initial therapy, exemplified by the medicinal composition described in [5] and its use.

Here, in the present invention, when an injection with foscarnet or the like as an active ingredient is used as a maintenance therapy after the first treatment of AD, for the purpose of preventing the reactivation of each virus, for example, the effective amount of foscarnet is set to 60~ 120 mg/kg/day, via intravenous injection, intramuscular injection or subcutaneous injection. It is expected to be intravenous drip for 2 to 6 months, and it is expected to be continuously administered for 2 to 3 months.

In addition, in order to check the reduction in the amount of viral DNA and its maintenance status, the recovery status of the patient’s cognitive function, and to prevent or regulate the maintenance of foscarnet tolerance or the occurrence of serious side effects such as renal impairment, the maintenance therapy can be appropriately adjusted The period and the dosage of foscarnet per time or per day.

[8] The aforementioned pharmaceutical composition contains an oral agent containing foscarnet as an active ingredient, and it is used together with/or replaced with maintenance therapy via injections, as the effective amount of foscarnet per day from 2000 mg to 6000 mg, divided into The medicinal composition for maintenance therapy characterized by being continuously administered 3 to 5 times a day for 3 to 6 periods can exemplify the medicinal composition described in [5] above and its use.

Here, when the oral preparation of the present invention using foscarnet as an active ingredient is used as maintenance therapy, it is used as a maintenance therapy via injections together/or replaced, and the effective amount per day of foscarnet is 2000 mg to 6000 mg. , Divided into 3 to 5 times a day, 3 to 6 periods of continuous injection. Furthermore, because foscarnet has mucosal irritation, it is expected to be combined with teprenone, sucralfate, azulene sulfonate sodium, and reba when administered orally. Rebamipide, polaprezinc, irsogladine maleate, benexate hydrochloride ( benexate ), sofalcone ( sofalcone ), cetraxate hydrochloride (cetraxate) , Ecabet sodium hydrate and other drugs with gastric mucosal protective effect are used in combination or form a mixture with these.

In addition, in order to check the reduction in the amount of viral DNA and its maintenance status, the recovery status of the patient’s cognitive function, and to prevent or regulate the maintenance of foscarnet tolerance or the occurrence of serious side effects such as renal impairment, the maintenance therapy can be appropriately adjusted Period and effective amount of foscarnet per dose or per day.

[9] The pharmaceutical composition described in [5] and its use are administered in combination with at least one preparation selected from the group of: ganciclovir, valacyclovir, penciclovir At least one nucleoside analog selected from the group consisting of penciclovir and brivudine; preparations of cidovir or its prodrugs, derivatives or other nucleotide analogs; preparations of nucleoside compounds ; Preparations of non-nucleoside DNA polymerase inhibiting compounds; preparations of amonevir or other helicase/initiating enzyme hindering compounds; packing hindering compounds for viral DNA capsid; letermovir or others Inhibitor of viral DNA terminal enzyme complex; a preparation containing nivolumab or other PD-1 antibodies and PD-1 receptor antibodies.

Here, the pharmaceutical composition for the treatment (including prevention) of AD using foscarnet as an active ingredient of the present invention may have antiviral effects against viruses that cause central nervous system diseases such as encephalitis that are different in chemical or mechanism of action. Compound preparations are administered in combination. At this time, you can vote at the same time or separately.

Preparations of compounds that can be used in combination include preparations of nucleoside analogs such as ganciclovir, valacyclovir, penciclovir, and brivudine, and nucleotide analogs such as cidovir or its prodrugs and derivatives. Preparations of substances, preparations of nucleoside compounds, preparations of non-nucleoside DNA polymerase inhibitor compounds, preparations of helicase/initiating enzyme inhibitor compounds such as Amonavir, packaging inhibitors of viral DNA capsid compounds, one of Letvvir method Inhibitors of viral DNA terminal enzyme complexes, preparations containing nivolumab-based PD-1 antibodies and PD-1 receptor antibodies, but are not limited to these.

[10] The pharmaceutical composition described in [5] and its use, which are used in combination with at least one preparation selected from the group of: donepezil, galantamine, and rivaroxil Ming (rivastigmine) or memantine (memantine) preparation, neuroprotective agent, neurotransmitter, at least one AD therapeutic agent or neurotransmitter mobility modifier selected from the group consisting of amyloid β or Aβ oligomer Targeted drugs; Tau protein as target drugs; inhibitors of excessive calcium influx into cells; vaccines, ibuprofen, ginkgo extract, or other anti-inflammatory agents; insulin or other anti-diabetic drugs; anti-IL-6 Antibodies, anti-IL-12 antibodies, or other inflammatory cytokines, chemokine neutralizers.

Here, the injection or oral agent (including modifiers and inhalants) for the treatment (including prevention) of AD using foscarnet as an active ingredient of the present invention can also be other AD therapeutics that have a different mechanism of action from foscarnet (" AD therapeutic agent") is administered in combination.

The AD therapeutic agents used in combination include, in addition to the preparations of donepezil, galantamine, rivastigmine, and memantine, neuroprotective agents, neurotransmitter substances or modifiers thereof, and amyloid beta (including oligomeric agents). Drugs) as target drugs, Tau protein as target drugs, inhibitors of excessive calcium influx in cells, vaccines, anti-inflammatory agents in the form of ibuprofen or Ginkgo extract, anti-diabetics in the form of insulin, and anti-IL- 6. Inflammatory cytokines such as antibodies and anti-IL-12 antibodies, and chemokine neutralizing agents, but are not limited to these.

[11] Diagnostic drugs for confirming the diagnosis of people at risk of AD or the progress of AD, or the therapeutic effect of antiviral agents used in the treatment of AD, including cerebrospinal fluid, plasma, blood, or saliva The specific viral DNA of HHV-6A, HHV-6B and HHV-7 in HHV-6A and HHV-7 are used as biomarker diagnostic drugs and the possibility of their use.

According to the present invention, AD is the first infection of "HHV-6" and "HHV-7" in infants and young children. Some people cause mild inflammation (HHV-6 encephalitis, tooth fever), but in the early stage That is, it is cured without becoming a serious problem with the passage of time.

After the initial infection, the "HHV-6" that is latently infected by the cells of the hippocampus or the limbic system of the brain is reactivated by reactivation with aging and decreased immunity. It has become clear that HHV-6-like encephalitis is a disease. Therefore, the use of specific viral DNA in the cerebrospinal fluid or blood, plasma, and saliva, respectively, specifically specific for HHV-6A, HHV-6B, and HHV-7, can confirm the risk of AD Diagnosis of people or the progress of AD and the therapeutic effect of antiviral agents used in AD treatment. At this time, the combined use of biomarkers or diagnostic methods related to AD such as amyloid PET, Tau PET, MRI, etc., enables more reliable diagnosis.

At present, there is no fundamental treatment drug developed to prevent or restore the progression of AD.

The first obstacles in AD are the hippocampus and its surrounding nerve tissues, which are responsible for the regeneration and division of nerve cells and early memory or cognitive functions. By inhibiting the proliferation of viruses in the hippocampus or surrounding tissues, the main cause of this disorder, it is possible to repair the nerve cell disorder with regenerative ability. As a result, not only can it be expected to prevent the progression of AD, but it is also possible to move toward recovery or cure.

The present invention is not only a symptomatic remedy for alleviating the symptoms of AD (especially memory/cognitive impairment), but is expected by preventing the reactivation of "HHV-6" and "HHV-7", one of the root causes of AD. The fundamental treatment or prevention of AD.

In summary, the increase in medical expenses for AD treatment can be suppressed, and moreover, the social burden and loss associated with AD suspension or care can be greatly reduced.

Figure 1 is a schematic diagram showing the Aβ hypothesis (cited based on Non-Patent Document 1). At present, the mechanism for the onset of AD has become the mainstream hypothesis.

Figure 2 is a diagram showing the outline of the "HHV-6 encephalitis cascade hypothesis regarding the onset of AD" (Aβ-related mechanism) and the point of action of multiple antiviral agents against this hypothesis (modified according to Non-Patent Document 1) .

Figure 3 is a graph showing a comparison between foscarnet and other antiviral agents, and is quoted from Figure 1 of Non-Patent Document 16. For HHV-6A, HHV-6B and HHV-7, compare the antiviral effects (EC) of foscarnet (PFA), acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), etc. _In the test of ₅₀ ), foscarnet was shown to be effective against these 3 viruses in the range of 30μg/mL. Furthermore, against HHV-6A and HHV-7, it was shown to be effective at about 1/2 the amount of HHV-6B. Compared with ACV, PCV and GCV, it has a balanced trend of obtaining efficacy.

Figure 4 is the second figure cited from Non-Patent Document 14. This is a graph showing the changes in plasma and cerebrospinal fluid concentrations of an aqueous solution containing 4000 mg of foscarnet 3 times a day for 3 days after oral administration to 6 AID patients. The plasma concentration reached a peak immediately after administration, was about 1/3 of the peak after 6 hours, and decreased to about 1/10 after 12 hours. However, in the cerebrospinal fluid, the peak value (approximately 25% of the plasma concentration) after administration for about 1 hour, then slowly decreases, after 6 hours it maintains more than 80% of the plasma concentration, and it rises back to the plasma after 12 hours concentration. Moreover, even for 12 hours, maintaining the IC ₅₀ concentration (50 μM/L) of HHV-6 (A and B) and HHV-7 can be guaranteed. (Quoted from "Raffi F, Antimicrob Agents Chemother 1993, 37(9)1777-80")

Figure 5 shows the relationship between the blood foscarnet concentration and the calcium ion concentration when the foscarnet preparation "Foscavir (registered trademark)" is administered to humans. Foscavir (registered trademark) (90mg/kg/12 hours as foscarnet) was confirmed to be negatively correlated between the concentration of foscarnet and calcium/ion concentration in the blood after repeated intravenous injection of Foscavir (registered trademark) in humans (r=0.96: p<0.001) The decrease of calcium ion concentration depends on the temporary change of dosage. After the instillation, the concentration of foscarnet decreases and slowly returns to the normal value. (Quoted from ``Foscavir (registered trademark) for intravenous drip injection 24mg/mL Interview Form March 2019 edition'')

Figure 6A is a graph showing the confirmation of β-secretase inhibition (N=1) through the concentration gradient study of Foscavir (registered trademark): the result of the enzyme inhibition test (Example 4) of BASE-1 by Foscavir (registered trademark). Foscavir (registered trademark) BASE-1 enzyme inhibitory activity When comparing the standard test antibody with the solution of Foscavir (registered trademark), the Foscavir (registered trademark) solution shows an inhibitory effect depending on the concentration, but its value is low .

Figure 6B shows the confirmation of β-secretase inhibition via the concentration gradient of Foscavir (registered trademark) (N=2).

Figure 7A shows the results of a test using a human neuroblastoma tumor strain ("SH-SY5Y strain") in the presence and absence of HHV-6/7 to study the effect of Aβ ₄₂ on cell proliferation (1). After 48 hours from the initiation of culture, due to the increase in the number of cells in any test group, no toxicity of extracellular Aβ ₄₂ to the test cells was observed.

Figure 7B shows the results of an experiment using a human neuroblastoma cell line ("SH-SY5Y strain") to study the effect of Aβ ₄₂ on cell proliferation in the presence and absence of HHV-6/7 (2).

Figure 8 is a graph showing the test results of using the SH-SY5Y strain to confirm whether Aβ ₄₂ is incorporated into cells in the presence of HHV-6/7. The Aβ ₄₂ addition conditions of 10 nM or less show the amount of intracellular Aβ ₄₂ that depends on the added concentration. Furthermore, even in the addition conditions of HHV-7 and HHV-6A virus solutions (group D), and the addition conditions of HHV-7 and HHV-6B virus solutions (group E), the amount of Aβ ₄₂ in the cells was also reduced tendency. These facts mean that the extracellular virus line inhibits the possibility of Aβ ₄₂ uptake into cells.

Further, in the group added foscarnet (A and F ~ H), when compared to the no addition group (A and C ~ E group), no clear difference in the amount of intracellular Aβ _42.

Fig. 9-1 is a graph showing the test results of studying the amount of viral DNA in the SH-SY5Y strain infected with the test virus. (Figure 9A and Figure 9B) HHV-6 was used as the test virus.

Fig. 9-2 is a graph showing the test results of studying the amount of viral DNA in the SH-SY5Y strain infected with the test virus. (Figure 9C and Figure 9D) HHV-7 was used as the test virus.

Figure 10 is a graph showing the test results of the study of the effect of HHV-6A on the human-derived lymphocyte strain (HSB-2 strain) in Example 5. Separately culture the unsupplemented group of virus solution (left picture) and HHV-6A virus solution added group (right picture), although the cells in (left picture) did not produce any changes, but the virus solution was added In addition (picture on the right), it was confirmed that there are cell-specific shapes during apoptosis (rough cells), cell-specific shapes during necrosis (swelling, etc.), unchanged cells, and fragments of dead cells. In the entire cell photographs in Figures 10 to 12, when compared with non-infected cells, although rough and uneven cells, cells with a large particle size, and dead cells are scattered among the virus-added cells, they are considered to be due to the virus. Changes in cells caused by infection.

Figure 11 is a graph showing the results of an experiment in which the effect of HHV-6B on the human-derived T cell strain (Sup-T1 strain) in Example 5 was investigated. Separately culturing the unsupplemented population of the virus solution (left image) and the HHV-6B virus solution added the population (right image), (left image) the test cells did not produce any changes. However, when each virus solution was added (right image), the cell-specific shape during apoptosis (rough cells), the cell-specific shape during necrosis (swelling, etc.), unchanged cells, and fragments of dead cells were confirmed.

Figure 12 is a graph showing the results of an experiment in which the effect of HHV-7 on the human-derived T cell strain (Sup-T1 strain) in Example 5 was investigated. Similar to Figs. 10 and 11, cells with a characteristic shape of cells at the time of multiple apoptosis or necrosis were observed.

Figure 13A is a graph showing the test results of the HSB-2 strain in Example 5 using anti-HHV-6A gp82 antibody to study whether it is infected with HHV-6A by immunostaining. Regarding the unadded group of HHV-6A virus solution (left picture) and the added group (right picture), when the presence or absence of virus infection is confirmed, there is no positive reaction due to antibodies (due to DAB staining). , (Right picture) most of the cells showed positive reaction.

Figure 13B is a graph showing the test results of the human-derived T cell strain (Sup-T1 strain) in Example 5 using an immunostaining method with anti-HHV-6B gp98 antibody to investigate whether it is infected with HHV-6B. When confirming the presence or absence of virus infection of the unadded group (left image) and the added group (right image) of the HHV-6B virus solution, there was no positive reaction due to antibodies (due to DAB staining). ), Yu (right) shows that most of the cells are positive. In addition, the infection test results of the immunostaining method shown in Figures 13B and 13C show that HHV-6B has a stronger infectivity to Sup-T1 cells than HHV-7.

Figure 13C is a diagram showing the test results of the human-derived T cell strain (Sup-T1 strain) in Example 5 using the HHV-7KR4 antibody immunostaining method to investigate whether it is infected with HHV-7. Confirm the unadded group of HHV-7 virus solution (left picture) and HHV-6B virus added group (right picture). When the infection is confirmed by immunostaining method, even if there is no antibody due to (left picture) A positive reaction (due to the dark brown staining of DAB), as shown in the right picture, shows that most cells are positive.

Figure 14 is a schematic diagram showing the course of AD onset according to the "HHV-6 encephalitis cascade hypothesis" of the onset of AD accompanied by the reactivation of latent infection of HHV-6/7 in the brain.

(2-1) Antiviral agents against HHV-6 (A and B) and/or HHV-7

The medical composition for the treatment and prevention of Alzheimer's disease (AD) of the present invention is a compound having antiviral effects on HHV-6 (A and B) and HHV-7 or a pharmaceutical composition (preparation) thereof Is effective

These compounds, as preparations, include foscarnet (Special Table Ping No. 10-509704, Special Table Ping No. 11-509515, Special Publication No. 2007-284452), Famciclovir (Special Publication No. 04-275229), Acyclovir ( U.S. Patent No. 49579244), Valaciclovir (Japanese Patent No. 3350055), Ganciclovir (Japanese Patent Laid-Open No. 08-53452), phosphonate derivatives containing cidovir (Japanese Patent No. 5963787) ), anti-CD40 antibody (Japanese Special Publication No. 2002-543150), pyridoquinoxaline (Japanese Special Publication No. 2005-516957), pyrazolopyridine derivatives (Japanese Special Publication No. 2004-529119), SITH-1 Inhibitors (Japanese Re-listed No. 2009-041501), nano/emulsified composition (Japanese Patent Application Publication No. 2011-518184), leptin derivatives (Japanese Patent Application Publication No. 2018-138586), etc. Among these compounds, foscarnet and ganciclovir are also applicable and effective for HHV-6 encephalitis, and are preferably used as active ingredients of medical compositions for the treatment and prevention of AD. Particularly better Is foscarnet which is known as a multiple antiviral agent. In addition, these compounds may be used in combination of two or more kinds, and used as a mixture or combination agent.

(2-2) About foscarnet

When "phosphonic acid" is mentioned in the present invention, it means the phosphonic acid represented by the following (Formula 1) or a salt thereof, or a solvate thereof. Here, as a salt, as a salt acceptable in medicine, metal salts such as lithium salt, sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt, methylammonium salt, dimethylammonium salt, and trimethylammonium salt , Dicyclohexyl ammonium salt and other ammonium salts, etc., can form hydrochloride, bromate, sulfate, nitrate, phosphate, metaphosphate and other mineral salts, methanesulfonate, benzenesulfonate, para Tosylate, acetate, propionate, tartrate, fumarate, maleate, malate, oxalate, succinate, citrate, benzoate, mandelate, cinnamon Acid salt, lactate, benzene sulfonate, valerate, stearate, oleate, gluconate, laurate, salicylate, theochlorate, tannate, butyl sulfonate Organic acid salts such as acid salts. As a solvent which can form a solvate acid, methanol, ethanol, isopropanol, acetone, ethyl acetate, dichloromethane, diisopropyl ether, etc. are mentioned.

The best compound among them is foscarnet sodium hydrate represented by the following (Formula 2), which is labeled Foscarnet Sodium in the (JNN)/INN label. Hereinafter, although the compound of (Formula 2) is mainly described as a typical "phosphonic acid", it is not limited to these.

Foscarnet is a compound that has strong antiviral effects on HHV-6 and HHV-7, and has excellent clinical effects on HHV-6 encephalitis after hematopoietic stem cell and organ transplantation. It has the following characteristics and is most suitable for It is an antiviral agent for the treatment (including prevention) of Alzheimer's disease.

(a) Antiviral effect

The effective amount for HHV-6A and HHV-7 is about twice as effective for HHV-6B, and the dosage of each virus does not need to be changed greatly, and it has an excellent balanced and strong antiviral effect. Furthermore, for HSV-1 and CMV, the effective amount of HHV-6A, HHV-6B, and HHV-7 also has a strong antiviral effect (Figure 3).

(b) Inhibition of β-secretase

Amyloid β (Aβ) is produced by decomposing and producing amyloid precursor protein (APP) by two types of amino acid degrading enzymes, β-secretase and γ-secretase.

In the present invention, it was confirmed that the foscarnet system has a low effect of inhibiting the enzyme activity of β secretase and/or γ secretase (Table 4), and the effect of inhibiting the production of Aβ can be expected.

(c) Inhibition of accumulation of amyloid β and Tau protein

In addition to the effect of (b) above, as long as the antiviral effect inhibits the proliferation of the virus, it can prevent the excessive production or secretion of Aβ, and its oligomerization. Not only this, it may also inhibit the accumulation of Aβ oligomers. Activation of excess phosphorylation of Tau protein occurs.

In order to confirm the above, Aβ ₄₂ , a mixture of HHV-6A and HHV-7, a mixture of HHV-6B and HHV-7, a single solution of HHV-7, and Foscavir were added to the medium of the SH-SY5Y strain (test cell) (Registered trademark) (800 μM), used to verify the uptake of Aβ ₄₂ in the test cell to measure its intracellular concentration, and to verify the uptake (infection) of the virus to measure the number of viral DNA.

As a result, compared with the cell population without addition of the virus solution of the comparative control, the amount of Aβ ₄₂ in the cells of the virus solution added population showed a tendency to decrease, and this decrease trend was not comparable to that of the virus solution added population with Foscavir (registered trademark) added. Differences (Figures 7 and 8). Furthermore, the number of HHV-6 DNA in cells in the Foscavir supplemented population decreased with the increase of Aβ. This line shows that the extracellular Aβ ₄₂ line under the test conditions is taken up into the test cell, the Aβ ₄₂ line inhibits the intracellular uptake of each virus tested, and the addition of Foscavir does not hinder the related inhibition.

The above results can be described as corroborative support to capture the natural immune effect of Aβ in viruses and other pathogens that appear outside the cell (W Eimer et al., Neuron 99(1): 56-63, 2018, RD Moir et al., Alzheimer's & Dementias 14 (2018): 1602-1614,201) is also applicable to HHV-6/7 infection. Furthermore, although the addition of Foscavir (registered trademark) shows the effect of reducing the number of viral DNA in the cell, it is due to the antiviral effect of Foscavir (registered trademark) or due to the ingestion of "virus Aβ complex" in cells Inhibition cannot be confirmed in this test condition. However, as a result of an experiment in which extracellular Aβ is taken up into cells in a concentration-dependent manner (additional conditions of 10 nM or less), there is a possibility that the intracellular uptake of "virus Aβ complex" may be suppressed.

(d) Inhibition of nerve cell death

Up to now, the cell line infected with HHV-6/7 undergoes apoptosis (R Ichimi et al., Journal of Medical Virology, 58(1): 63-68, 1999, and non-patent literature 10, reference 30, 31). In the above-mentioned test, the presence or absence of death of infected cells was tested by the related viruses. (Example 5)

The HHV-6A obtained in Example 1, the uninfected HSB-2 strain (human T lymphoblastoid cell strain), the uninfected Sup-T1 strain of HHV-6B and HHV-7 (derived from human T cell strains of T lymphocytes) each virus solution was added to observe the presence or absence of virus infection and the morphological changes of the infected cells (Example 5).

As a result, in the uninfected cell population of HHV-6A (Fig. 10-1), the shape or size of each cell was uniform, and almost no dead cells were observed. However, in the infected cell population (Figure 10-2), hypertrophic cells and dead cells were scattered.

Furthermore, with regard to HHV-6B and HHV-7, in the uninfected Sup-T1 population (Figure 11-1), no dead cells were seen, and the shape and size of the cells were also uniform. However, in the HHV-6B-infected cell population (Figure 11-2) and HHV-7-infected cell population (Figure 12), in addition to dead cells, the cell surface was scattered with rough cells and hypertrophic cells.

Through the above test, in the cells infected with HHV-6A, HHV-6B, and HHV-7, it is obvious that the cell surface has unevenness and hypertrophic morphological changes. Among them, the size of the cells with bumps on the cell surface does not change from that of the non-infected cells. As the cell apoptosis is based on changes in the morphology of the cell membrane as the starting point, it is shown as apoptotic cells.

On the other hand, since the hypertrophy of the cell becomes a typical form of necrosis (Reference 24), the hypertroped cell is believed to be a necrotic cell. Furthermore, although there are many cells showing a positive reaction by immunostaining, the number of cells undergoing apoptosis and necrosis is few, and it is clear that most of the infected cells show a normal state (morphology).

Through the above-mentioned experiments, cells infected with HHV-6A, HHV-6B, and HHV-7 not only apoptosis, but also cause necrosis for the first time.

The above findings mean that in the brain of AD, especially in the hippocampal gyrus where neuronal cell regeneration occurs, HHV-6A or HHV-6B that is latently infected with nerve cells or glial cells is involved in the apoptosis of host cells. Reactivation at the time of cell death also causes necrosis. The cell causes various inflammation-causing substances (such as HMGB-1, TDP-43, GSK-3β, glutamine, glutamine, etc.) as DAMPs (damage/harm related molecular forms). Ca ^++, etc.) are released to increase inflammation.

(e) Security

Foscavir (registered trademark) is a drug with a wide safety range in terms of the effective dose and toxic dose of HHV-6A, HHV-6B and HHV-7 as the target (Figure 3). For CMV infection, it has been recognized for clinical use in Japan, the United States, Europe and other countries around the world for a long time (Non-Patent Document 13), so it can be safely used in the treatment of AD.

(f) Treatment effect on AD

In the present invention, since the extracellular Aβ system is confirmed to be taken into the cell regardless of the presence or absence of virus (Example 1), as long as Aβ has its own immune response to viral infection, it is not limited to inhibit viral infection, and the production of Aβ is not limited. If it stops, there is a high possibility that the secreted Aβ is taken into the cell and accumulated. Since the onset of AD is related to the pathological effects of Aβ, the treatment of AD can be expected by inhibiting the reactivation of the cause of Aβ in the brain, especially the latent HHV-6/7 in the hippocampus.

Although it is believed that the main affected area of AD is the hippocampal gyrus and its surrounding nerve tissue, its condition can be explained as the reactivation of HHV-6/7 in the hippocampal gyrus and its surrounding nerve tissue, whereby Aβ accumulates, through infection with virus or migration to The process of neurological breakdown (AD/"HHV-6 encephalitis cascade hypothesis") caused by pathological development such as cell damage to T cells at the inflammatory site. Furthermore, because the neurological function of the hippocampal gyrus has the ability to regenerate, it is expected that the dead cells will be filled or the obstacles will be repaired. Therefore, if the antiviral agent having the anti-HHV-6/7 action of the present invention such as foscarnet is used for early stage Treatment can be expected asymptomatic. Furthermore, as long as the neural stem cell layer that exists in the hippocampal dentate gyrus does not collapse, the possibility of recovery can also be expected in severe AD.

Activated T cells that migrate to the site of infection usually die due to apoptosis and have no effect when fulfilling their role. However, in the present invention, in addition to apoptosis, it is confirmed that it also causes the release of DAMPs and other necrosis (Example 5). Therefore, in the prevention of AD or its treatment, it is indispensable to exclude factors that impart migration such as T cells, and the antiviral agent that inhibits the reactivation of HHV-6/7, which is considered to be the factor, is Suitable for.

In addition, patients with HHV-6 encephalitis ("post-transplant HHV-6 encephalitis patients") who are symptomatic after transplantation of hematopoietic stem cells or organs show cognitive impairment similar to those observed in patients with AD menstrual disorder Neurological symptoms, as the most suitable method/dosage of antiviral agents with anti-HHV-6/7 effects for AD patients, refer to the use/dosage of confirmed significant therapeutic effects for patients with HHV-6 encephalitis after transplantation. For example, in the case of foscarnet, excellent treatment results have been reported in the case of HHV-6 encephalitis patients with a full dose (180 mg/kg/day) intravenously administered intravenously over several days.

Furthermore, it is known that foscarnet has the effect of binding to Ca ⁺⁺ , and clinically has side effects such as hypocalcemia. Conversely, it can be expected that the excess cells inside and outside give Ca ⁺⁺ reduction and restoration effects (Figure 5, 5). In particular, Aβ has an effect on the influx of excess calcium in cells by increasing calcium channels (A Drews et al., Scientific Reports 6:31910:1-12,2016), the cause of Aβ production is HHV-6/7 Inhibition of reactivation indirectly confers a decrease in intracellular Ca ⁺⁺ .

In addition, foscarnet has an antiviral effect on reactivated HHV-6/7 in the brain, and it is necessary to reach the infected site through the BBB. Since foscarnet passes through the BBB, it can be maintained for about 6 hours after being administered in the cerebrospinal fluid at an effective concentration for HHV-6/7 (Figure 4), which is suitable for AD treatment.

It can be seen from the above that the administration of antiviral agents for HHV-6/7 inhibits at least the production of Aβ, the uptake of Aβ into human nerve cells, the formation of oligomers, and/or the accumulation of Aβ. AD is possible by treatment with antiviral agents that are effective against HHV-6/7.

The main participant of HHV-6 encephalitis is HHV-6B. In the case of AD, it is known that the reactivation of HHV-6A and HHV-7 is likely to be related to the expansion and increase of the disease, and it is caused by HSV-1. Or encephalitis caused by cytomegalovirus (CMV) infection has dementia-like symptoms, and the reactivation of these other herpes viruses also promotes the secretion of Aβ, which may also become the main cause of AD.

Therefore, antiviral agents targeted for AD treatment not only have an effect on HHV-6, but also have an effect on HHV-6A and HHV-7. It is further expected to have multiple antiviral effects on other herpes viruses such as HSV-1 or CMV. Since foscarnet has such multiple antiviral activities, it is expected to have a therapeutic effect on AD.

<Embodiments of the antiviral agent of the present invention for the treatment and prevention of AD>

The present invention relates to the use of antiviral agents having excellent antiviral effects against human herpesvirus types 6 (HHV-6A and HHV-6B) and type 7 (HHV-7), etc., for the treatment and prevention of AD Although the use includes the following first to eleventh embodiments, it is not limited to these.

The first form of antiviral agent that can be used for the treatment (including prevention) of Alzheimer’s disease (AD) will be effective against human herpesvirus type 6 (HHV-6A and/or HHV-6B) and/or human herpes Herpes virus type 7 (HHV-7) has a strong antiviral effect as an effective ingredient drug, and it is expected to be effective against herpes simplex virus type 1 (HSV-1), cytomegalovirus (CMV), etc. Compounds with multiple antiviral effects are used as active ingredients.

The second form is the above-mentioned compound with antiviral effect. It is a compound that inhibits the DNA polymerase of herpes virus, a helicase/initiator complex compound required for the synthesis of viral DNA-6, and it inhibits the packaging of viral DNA capsid Compounds and pyrophosphate analogs that directly bind to the pyrophosphate of viral DNA polymerase and selectively inhibit DNA synthesis. Among them, pyrophosphate analogues (phosphoric acid analogue compounds) do not need to be activated by phosphorylase, have a low possibility of resistance, and are scheduled to be administered for a long time and are suitable for AD treatment.

Compounds with antiviral effects other than pyrophosphate analogs can also be expected to selectively inhibit the growth of viruses such as viral DNA polymerase. Especially when combined with pyrophosphate analogs, it can complement the antiviral effect of pyrophosphate analogs.

The third aspect is that the above-mentioned pyrophosphate analog is phosphonoacetic acid or phosphonocarboxylic acid and/or derivatives thereof, which bind to the pyrophosphate binding site of DNA polymerase, and can be expected to selectively inhibit virus proliferation activity. Or calcium antagonism and anti-aspartic acid protease effect. In addition, the phosphonoformic acid represented by the following basic skeleton (Formula 1), or a salt thereof, or a solvate thereof, etc. is referred to as a "phosphinylformic acid derivative".

The fourth aspect of the phosphonoformic acid derivative is a compound represented by the following (Formula 2) of the molecular formula "CNa ₃ O ₅ P/6H ₂ O". Although not limited to the present inventor, the compound represented by (Formula 2) was used as the "phosphonic acid" in this example.

The above-mentioned foscarnet preparations (injections) are sold under the trade names of "Foscavir (registered trademark)" (Japan) or "Foscavir" (U.S., etc.) mainly for cytomegalovirus infection indications in various countries.

The fifth aspect is a method of using a preparation containing foscarnet as an active ingredient and an oral preparation for the treatment (including prevention) of AD.

This use includes the treatment of preclinical AD. Preclinical AD refers to the state from the stage where the accumulation of amyloid β in the brain is proven to before the initial cognitive impairment (MCI), including Stage 1 is the stage where the accumulation of amyloid β in the brain is observed. 2 is the stage where the accumulation of amyloid β is accompanied by the discovery of variability in the brain, and Stage 3 is the third stage in which the insignificant cognitive function decline stage except for Stage 2. For the treatment of preclinical AD, Stage 3 is expected, and Stage 2 is expected to start.

The sixth form is a method that uses a foscarnet preparation for AD treatment as an injection, and its use is also for the purpose of pre-emptive therapy. The initial therapy system significantly reduces the amount of each viral DNA of HHV-6A, HHV-6B and HHV-7 in the cerebrospinal fluid and/or plasma or blood. It is desired to reduce it to below the level of a healthy person, and it is more desirable to reduce it to below the detection level for the purpose of allowing AD patients admitted to the hospital, for example, to divide the effective dose (60~180mg/kg/day) of foscarnet into 1 2 or 3 times a day, via intramedullary injection or intravenous injection over a period of 3 to 10 days, desirably a method of continuous administration for 3 to 5 days.

Furthermore, the method for checking the DNA of each virus may be a method specified in Non-Patent Document 3, or a method commonly used by other medical institutions. Furthermore, the amount of each viral DNA targeted for reduction may be appropriately corrected in consideration of the patient's condition.

Furthermore, in order to consider the reduction in the amount of DNA of each virus and its maintenance status, the recovery status of the patient's cognitive function, and the prevention or regulation of the maintenance of foscarnet tolerance or the occurrence of serious side effects such as renal impairment, the period of the first treatment The dosage of foscarnet and foscarnet can be adjusted appropriately every time or every day. Furthermore, with regard to intramedullary administration, since the agent can migrate to the site of direct infection, the dosage can be further reduced.

The seventh form uses foscarnet preparations as injections, as a maintenance therapy after the first treatment, for the purpose of preventing the reactivation of each virus, for example, 60~ 120mg/kg/day is divided into 2 to 3 times a day, intravenously, intramuscularly, or subcutaneously. It is expected to be intravenously injected, and the method of administration is 3 to 6 months.

In addition, in order to check the reduction in the amount of viral DNA and its maintenance status, the recovery status of the patient’s cognitive function, and to prevent or regulate the maintenance of foscarnet tolerance or the occurrence of serious side effects such as renal impairment, the maintenance therapy can be appropriately adjusted Period and the dosage of foscarnet per 1 time or per day.

The eighth aspect is to use foscarnet preparations as oral preparations, together with or instead of maintenance therapy via injections. For example, the effective amount of foscarnet per day of 2000 mg to 6000 mg is divided into 3 to 5 times a day, 3 A maintenance treatment method characterized by continuous administration to 6 months. Because foscarnet has mucosal irritation, it is expected to be combined with teprenone, sucralfate, azulene sodium sulfonate, and rebamipide during oral administration. ), polaprezinc, irsogladine maleate, becenate hydrochloride, sofalcone, cetraxate hydrochloride, irsogladine maleate Ecabet sodium hydrate and other drugs with gastric mucosal protective effect are used in combination or form a mixture with these.

In addition, in order to check the reduction in the amount of viral DNA and its maintenance status, the recovery status of the patient’s cognitive function, and to prevent or regulate the maintenance of foscarnet tolerance or the occurrence of serious side effects such as renal impairment, the maintenance therapy can be appropriately adjusted Period and the dosage of foscarnet per 1 time or per day.

The ninth aspect is a method of using a compound that has an antiviral effect that is chemical or a different mechanism of action from the preparation of foscarnet for the treatment (including prevention) of AD, or a preparation containing the compound.

Concomitant antiviral agents include famciclovir (Japanese Patent Publication No. 04-275229), acyclovir (U.S. Patent No. 4,957,924), valacyclovir (Japanese Patent No. 3350055), valganciclovir (valganciclovir), Penciclovir, brivudine and other preparations containing nucleoside analogues, cidovir (Japanese Patent No. 5963787 Number), preparations containing nucleotide analogs such as prodrugs or derivatives thereof, or preparations containing nucleoside compounds, preparations containing non-nucleoside DNA polymerase inhibitory compounds, preparations containing helicase/initiating enzyme complex inhibitory compounds , Containing compounds that inhibit the packaging of viral DNA capsids, anti-CD40 antibody (Japanese Patent No. 2002-543150), PD-1 antibody (Japanese Patent No. 5701266), tetrahydro-2H-thiopyran carboxamide derivatives (Japanese Patent No. 5011739), but not limited to formulations of leptin derivatives (Japanese Patent Application Publication No. 2018-138586).

The tenth aspect is a foscarnet preparation for the treatment and prevention of AD that has a different mechanism of action for the purpose of AD treatment (AD therapeutic agent), specifically, aggregation of Aβ and Tau protein that are the cause of the deterioration of AD A compound that inhibits phosphorylation/neurofibrillary changes, or has a compound that directly acts on the production/transmission of neurotransmitters such as acetylcholine, or a method of using the compound as an active ingredient in combination.

Foscarnet suppresses the proliferation of viruses that cause excessive cell death, and therefore has the effect of suppressing the production or secretion of excessive Aβ that is a natural immune response.

Furthermore, the foscarnet system has the possibility of exerting an anti-aspartic protease effect in cells, and it can be expected to inhibit the production of Aβ. However, due to the inability to have a strong effect, in the treatment of AD, it has the effect of inhibiting the aggregation of Aβ, phosphorylation of Tau protein, or neurofibrillary changes, or the modulation of the production/transmission of neurotransmitters such as acetylcholine. It is useful to use a compound that acts directly or a drug containing the compound as an active ingredient.

As the drug to be used in combination, there are acetylcholinesterase-blocking drugs in the donepezil system, or galantamine-based drugs that have a nicotinic acid receptor enhancing effect together with acetylcholinesterase inhibition, and rivaroxil It is a drug that inhibits both acetylcholinesterase and butycholinesterase, and memantine is a drug that inhibits NMD receptors.

Furthermore, it may also interact with an agent that acts on the secretase that decomposes Aβ to inhibit the production of Aβ (Japanese Special Publication No. 2010-529014, Japanese Special Publication No. 2011-518224), or a promotion agent that hinders or breaks down the accumulation of Aβ, or promotes Aβ Drugs discharged from the ventricle, or drugs that inhibit the aggregation of Aβ or the production of Aβ fibrils (Japanese Special Form 2017-521427), or protein polymerization/activation inhibitors (Japanese Special Form 2003-528171, Japanese Special Form 2017 -521427) or oxidative stress/endoplasmic reticulum stress-induced apoptosis inhibitors (Japanese Patent Application Publication No. 2008-239538), vaccines (Japanese Patent Application Publication No. 2007-522119, Japanese Patent Application Publication No. 2010-522559), etc.

Further, it is combined with excessive calcium influx inhibitors, ibuprofen, ginkgo extract, or other anti-inflammatory agents, insulin or other anti-diabetics, anti-IL-6 antibodies, anti-IL-12 antibodies, or other The combined use of neutralizing agents of inflammatory cytokines and chemokines is also possible.

The eleventh form is used as a biomarker to confirm the effects of AD diagnosis and therapeutic drugs. HHV-6A, HHV-6B and HHV-7 viruses are detected from the saliva, blood, plasma, and cerebrospinal fluid (CSF) of AD patients Methods by itself, viral DNA or antibodies or fragments thereof. Among them, the most accurate method for confirming viral infections in the brain, especially the hippocampus, is to measure viral DNA in CSF.

At this time, as a biomarker for confirming the therapeutic effect of antiviral agents in the diagnosis and treatment of people at risk of AD or the progress of AD, it is used in the cerebrospinal fluid, plasma or blood of the subject , Or saliva, HHV-6A, HHV-6B, and HHV-7 respectively specific virus DNA, protein, glycolipid, or viral constituent glycoprotein, etc. in blood or saliva. In this case, it can be used in combination with amyloid PET, Tau PET, MRI and other diagnostic methods or biomarkers related to AD.

The biomarker for the method of confirming the risk diagnosis of AD, the diagnosis of the progress of AD, and the therapeutic effect of AD therapeutics including antiviral agents used in the treatment of AD is the starch-like substance in the universal CSF Protein, Tau protein, amyloid PET examination, fluorine-D-glucose PET, MRI examination, and moreover, various methods have been developed as biomarkers for confirming the diagnosis and treatment effect of AD.

However, it is currently unknown how to use viral DNA of HHV-6A, HHV-6B and HHV-7 as a biomarker for AD diagnosis.

Although we are developing a novel technology for the determination of viral DNA, we can use the "Pathogen Inspection Manual" (Reference 24) of the HHV-6 and HHV-7 viruses, which are the cause of sudden rash, prepared by the National Institute of Infectious Diseases. The prescribed real-time PCR method is used as the standard method.

The foscarnet preparation of the present invention is not only for the treatment of AD, but can also be used as a latent infection of herpes viruses, especially the reactivation and/or proliferation of HHV-6 and HHV-7, and the amyloid Aβ It is a therapeutic agent for the following diseases caused by accumulation and antiviral effect of foscarnet.

Treatment (including prevention) HHV-6 encephalitis (infants and young children), sudden rash, pneumonia, age-related macular degeneration, oral cancer, chronic fatigue syndrome, severe depression syndrome, bipolar disorder, heart failure, drug allergy , Idiopathic thrombocytopenia purpura, pityriasis rose, multiple sclerosis, Lewy body degeneration, spinal cord/cerebellar degeneration, meningitis, Hashimoto's disease, epilepsy, medial lateral cephalosclerosis, anterior head Lateral cephalic lobe degeneration, amyotrophic lateral sclerosis, Huntington's chorea, Rosemiao's encephalitis, autoimmune encephalitis, limbic encephalitis, HSV-1 encephalitis, CMV encephalitis, HIV encephalitis, EVB encephalitis, EKV nephropathy, hemorrhagic cystitis/interstitial nephritis/urethral stenosis after hematopoietic stem cell/bone marrow or organ transplantation.

The parenteral preparations of foscarnet of the present invention include injections, drips, packaging agents for drips, and nasal dosage forms for subcutaneous, intramuscular, intramedullary or intravenous administration.

For pre-emptive therapy, it is desirable to be a preparation for intravenous injection that is formulated to be administered in a high dose, and further, it is desirable to be a preparation for drip intravenous injection. In the case of injections, it can be prepared by dissolving foscarnet in an appropriate diluent (physiological saline, etc.), applying sterilization treatments such as filter sterilization, and filling them into sealed containers such as ampoules, vials, and infusion packages by a method known to those skilled in the art. manufacture.

In the case of foscarnet alone preparations for oral use in maintenance therapy, the preparations are made by general manufacturing methods with excipients, disintegrants, binders, lubricants, suspending agents, isotonic agents, and emulsification Additives such as agents, sweeteners, flavors, and coloring materials can be manufactured by mixing the foscarnic acid with the foscarnet by conventional methods.

Foscarnet is prone to chelate with metals such as compounds with extremely high stability. Because of its mucus irritation, it is expected that it is easily decomposed as an excipient such as starch, lactose, and mannitol.

Furthermore, for the purpose of accelerating or slowing the absorption from the intestinal tube, the foscarnet can be enclosed in microcapsules or made into a multi-structured lozenge by a conventional method.

When foscarnet is used as a formulation with a high dosage of more than 1 gram, it is difficult for the elderly to take the formulation when the formulation is usually large in the case of tablets or capsules (including soft capsules). At this time, granules, fine granules, powders or gel preparations can also be used.

In the case of oral liquid preparations such as drinks, use distilled water for injection or physiological saline as a base, dissolve foscarnet according to the usual method, add salt, sugar, syrup, etc. as needed, and fill it into a glass vial or PET Containers etc. can be manufactured.

In addition, because foscarnet has mucosal irritation, it is expected to be combined with teprenone, sclafil, sodium azulene sulfonate, rebamipide, polarizin, and isoladine during oral administration. Maleate, becenaide hydrochloride, sofadone, cetraxate hydrochloride, icafibrate sodium hydrate, and other agents with gastric mucosal protective effects are used in combination or formed into a mixture with these.

The combination of foscarnet and AD or gastric mucosal protective agent can be manufactured in the same way as the single foscarnet. In addition, microcapsules encapsulated with foscarnet and capsules (including nanotubes/capsules) encapsulated with AD agents or gastric mucosal protective agents can be mixed and manufactured.

Regarding the diagnosis of AD, according to symptoms, it is divided into three stages: initial (mild), intermediate (moderate), and late (high) stages. Existing AD treatment drugs are also adapted according to this stage.

Foscarnet can also be used in any stage of AD in terms of its mechanism of action. Although there are drugs suitable for the treatment of severe AD from the recommended dosage as a drip intravenous injection, based on the experience of the treatment of HHV-6 encephalitis, it is expected that the administration at the initial or mild stage will stop the progression of AD ,We are expecting a reply. This makes the use of foscarnet for preclinical AD possible.

Preclinical AD refers to the state of the accumulation of Aβ in the brain from the proven stage to before the initial cognitive impairment (MCI), including stage 1 is the accumulation stage of Aβ observed in the brain, and stage 2 is the accumulation of Aβ Along with the discovery of the degeneration of the brain, stage 3 is the third stage at the beginning of the insignificant cognitive decline except stage 2. For the treatment of pre-clinical AD, it is expected to start at stage 3, and more expected to start at stage 2. Foscarnet is also suitable for treatment at this time point.

As a biomarker for AD, the ratio of Aβ and Aβ ₄₂ to other Aβ in cerebrospinal fluid (CSF), Tau protein, amyloid PET examination, fluoro-D-glucose PET, MRI examination, etc. are widely used. However, in these methods, it is impossible to specify the presence or type of the virus which is the main cause of AD.

Although the technical evolution of the viral DNA method for HHV-6A, HHV-6B, and HHV-7 has been significant, the National Institute of Infectious Diseases can use the HHV-6 and HHV-7 viruses that are the cause of sudden rashes. The real-time PCR method specified in the "Pathogen Inspection Manual" (Reference 24) as the standard method.

The present invention aims to treat people with AD by inhibiting the reactivation of latent HHV-6/7. The DNA level of HHV-6/7 in the brain is focused on the cerebrospinal fluid (CSF), which is relatively low invasive and can be measured. The most sensitive inspection method is the real-time PCR method.

In antiviral therapy for AD, the degree of DNA of HHV-6/7 in CSF is used as an indicator of preventive and therapeutic effects.

It is most desirable that the viral DNA of HHV-6A, HHV-6B, and HHV-7 in the CSF of AD patients is below the detection limit. Since there are reports that the amount of DNA of HHV-6A and HHV-7 detected in the brain (parahippocampal gyrus, etc.) of AD patients is mainly about twice that of non-AD patients, it is expected to be at least the amount of DNA less than that of non-AD patients.

As the first treatment for HHV-6 encephalitis showing acute and severe symptoms, it has been reported that the highest dose (180mg/kg/day) administration of foscarnet in several days (the shortest is 3 days) can make the cerebrospinal fluid The DNA of HHV-6B is below the detection limit.

However, the condition of AD is indeed common to HHV-6 encephalitis in terms of symptoms of cognitive decline, but is different from HHV-6/7 high-concentration infection/highly active HHV-6 encephalitis, and it is considered to be stable get on. Therefore, the dosage of foscarnet in AD treatment is also highly likely to be set fairly stably.

The latent form of HHV-6 cannot completely inhibit its reactivation. After the initial therapy has reached the level of healthy people or the viral DNA in the cerebrospinal fluid is below the detection limit, switch to maintenance therapy, and you can also choose to increase the patient's medically prescribed dosage as a preparation method.

In this specification, the "effective amount" is the amount necessary to prevent the infection degree of HHV-6/7 in the cells of the cranial nerve system from at least increasing. The degree of infection can be regarded as the HHV-6/7-derived in CSF. Evaluator of the degree of DNA accumulation. The specific dosage varies according to the patient's viral infection status or symptoms, physical conditions, route of administration, and the efficacy of antiviral agents against each virus.

In the treatment of foscarnet preparations for AD, it is desired to maintain the level of infection of HHV-6A, HHV-6B, and HHV-7 in the brain at an early stage below the level of healthy people or the detection level.

As mentioned above, in the indications of foscarnet for HHV-6 encephalitis, the full dose (180mg/kg/day) after the onset of intravenous injection shows excellent effects. For example, for AD patients, when they are hospitalized A 250 mL vial (24 mg/mL) injection ("Foscavir (registered trademark) Note" preparation) containing 6 g of foscarnet sodium hydrate as the active ingredient was used as a first-in-class therapy.

Although the maximum amount of the effective dose for the initial administration of the pre-emptive therapy is based on 180 mg/kg/day, the effective dose is appropriate for patients with complicated renal disorders or for intramedullary administration in order to follow the doctor’s instructions. Fix. As an effective amount during the hospital stay of maintenance therapy, dissolve "Foscavir (registered trademark) Note" in 1000 mL of normal saline, and administer 125 to 250 mL (0.75 to 1.5 g as foscarnet) once. . Furthermore, depending on the situation, intravenous injection or drip intravenous injection can also be considered.

Furthermore, for example, an oral preparation containing 500 mg to 1500 mg as foscarnet is prepared and used as maintenance therapy after discharge. In continuous therapy, 2000-6000 mg foscarnet is administered in 3 to 4 times a day. This period is until AD symptoms (especially cognitive impairment) disappear, and the goal is 3 to 6 months. Furthermore, because foscarnet has mucosal irritation, it is expected that it should be combined with teprenone, sclafil, sodium azulene sulfonate, rebamipide, bolarizine, and isoladine once the oral agent is administered. Gastric mucosal protective agents such as maleate, becenate hydrochloride, sofadone, cetraxate hydrochloride, and icafibrate sodium hydrate are used in combination or form a mixture with these.

In addition, the DNA amount of HHV-6A, HHV-6B, and HHV-7 in the cerebrospinal fluid, plasma or saliva is detected regularly (once in 1 to 3 months). If the amount of DNA exceeds the benchmark, it will be hospitalized again. The administration of foscarnet was performed according to the pre-emptive therapy. The dosage is appropriately adjusted according to the state of the patient or the amount of virus detected.

The AD system occurs in the elderly, and most of the elderly have decreased renal function. In the case of high-dose administration, it is desirable to reduce the risk of renal dysfunction through diuretics or sufficient water supply after administration.

Furthermore, in consideration of renal dysfunction, in addition to reducing the dosage by intramedullary administration, it is desirable to change the dosage of foscarnet based on the value of urine creatine. The method for changing the dosage is routine, and its details are described in the treatment guidelines for HHV-6 encephalitis (Ref. 32). This guideline can also be applied to foscarnet therapy for AD.

[Example]

Although examples are shown below to specifically explain the present invention, the present invention is not limited to these.

The other terms or concepts in the present invention are based on the meanings of terms used in the technical field, and are used to implement the various technologies used in the present invention. In particular, in addition to the technology that clearly indicates the source, as long as it is Those skilled in the art can implement it easily and reliably based on known documents and the like. In addition, various analyses, etc., used analytical equipment or reagents, are performed in accordance with the methods described in the kit's operating instructions and catalogs.

In addition, the descriptions in the technical documents, patent gazettes, and patent application specifications cited in this specification are referred to as the descriptions of the present invention.

(Reference Example 1-1) The effect of foscarnet on HHV-6-1

HHV-6 was inoculated into the human T lymphocyte cell line HSB-2 (ATCC), and after the infection was established, trisodium foscarnet hexahydrate (hereinafter referred to as "foscarnet" in this reference example) was added. References below The examples and examples are also the same.) 67 μM cultured for 14 days. Count the number of live cells, and count the HHV-6 antigen-positive cells by fluorescent antibody method.

The results are as shown below. Foscarnet completely inhibited the cell disorder caused by HHV-6 and the realization of viral antigen-positive cells at 67 μM.

(Reference example 1-2), the effect of foscarnet against HHV-6-2

Foscarnet and other widely used herpes virus agents (Acyclovir, Penciclovir, Ganciclovir) against HHV-6 are based on the source of use A comparison of human T-lymphoblast cells and cord blood lymphocytes has reported results as described below (Non-Patent Document 17).

Compared with other antiviral agents, foscarnet has extremely high anti-HHV-6 activity, and the difference between the effective amount and the toxic amount is the largest, and the safety is excellent.

(Reference example 2) Comparison of foscarnet against HHV-6A and HHV-6B and other agents

When comparing the effect of foscarnet against HHV-6A and HHV-6B with other antiviral agents, the human T lymphoblastoma cell line HSB-2 cells (ATCC) and Molt-3 cells (ABI) were respectively infected HHV-6A GS strain (NIH) or HHV-6B Z29 strain (ABI company) for antiviral analysis and cytotoxicity analysis. That is, based on the analysis of the antiviral respective cells per ¹⁰⁶ infected cells it is HHV-6 100CCID _50, by adding to the 10% FCS, 2mM L- bran Amides, RPMI culture medium with 0.1% sodium bicarbonate , Let it absorb for 90 minutes at 37°C. After washing and adjusting the number of cells, the adjusted antiviral agents are serially diluted and added. Antiviral agents added to every 3 to 4 by the medium exchange at 10 to 12 was calculated to be inhibited virus proliferation in a concentration of up to 50% of the antiviral agent as CC _50. (Non-Patent Document 16)

(Reference Example 3) The effect of foscarnet and other antiviral agents against HHV-7

The effect of foscarnet against HHV-7 was obtained by using a human-derived Sup-T1 cell line and purified CD4+ T lymphocytes. The results of comparison with other antiviral agents are as follows (Non-Patent Document 17).

As shown above, the antiviral effect of foscarnet against HHV-6B is about twice the effective amount against HHV-6A and HHV-7. The dosage of each target virus does not need to be increased, which is a balanced Antiviral agents (Figure 3).

(Reference Example 4) Anti-multiple virus activity of foscarnet

The foscarnet system has antiviral effects against various viruses as described below (Non-Patent Document 17). In particular, CMV, HSV-1, EBV, HIV-1, etc. are known to be latent infections in the brain, and when activated, they are known to cause encephalitis. Therefore, as an antiviral agent for AD therapeutics, it is expected to have an effect against related viruses (Clinigene Co., Ltd., "Foscavir (registered trademark)/Interview Form March 2019 Edition", 2019).

(Reference Example 5) The brain migration of foscarnet

Transition of Foscarnet on Cerebrospinal Fluid

When AIDS patients (n=27) inject 56~213mg/kg of foscarnet (median value: 100mg/kg) intravenously for 2 to 6 hours, the concentration of foscarnet in the cerebrospinal fluid is 50~250nmol/ mL, equivalent to 10 to 70% of the plasma concentration. Six hours after the administration, the concentration in the cerebrospinal fluid was almost the same as the concentration in the blood. (Non-Patent Document 15, Figure 4)

(Reference Example 6) The effect of foscarnet on calcium

(6-1) The effect of foscarnet on free calcium-involving the effect of ganglion junction

Rats were used to remove diaphragmatic ganglion specimens to review the effect of foscarnet on contraction induced by electrical stimulation. Furthermore, the concentration of free calcium in the nutrient solution when foscarnet was added was measured. As a result, foscarnet did not involve the influence from 0.1 mM to 3 mM. Although an average 14% contraction inhibition was observed after 10 minutes at 10 mM, there was no statistically significant difference. At the end of the experiment, the Ca ⁺⁺ ion concentration in the nutrient solution containing 10 mM foscarnet was reduced by 7% compared with the control.

Above, in the diaphragm ganglion specimens extracted from rats, foscarnet at 10 mM showed a tendency to inhibit contraction induced by electrical stimulation.

(6-2) The effect of foscarnet on imparting calcium stability

When foscarnet 405mg/kg was administered intravenously to dogs, the total serum calcium value decreased from 2.5mmol/L to 2.0mml/L, and the free calcium concentration decreased from 1.3mmol/L to 0.9mmol/L. At this time, in 1/3 cases, the head trembles and the neck muscles become tense. In the administration of 810 mg/kg, the total serum calcium value decreased from 2.5 mmol/L to 1.6 mmol/L, the free calcium concentration decreased from 1.3 mmol/L to 0.7 mmol/L, and muscle contracture was observed.

Moreover, symptoms such as the decrease of free calcium concentration and muscle contracture disappeared within 24 hours after the end of the administration.

In a human CMV omentitis patient, a single dose of foscarnet was administered at 90 and 120 mg/kg intravenously, and the dose-dependent reduction in serum calcium was confirmed (Figure 5). At this time, 2 out of 11 patients who had been administered 120 mg/kg were observed to have irritation and pain around the mouth and numbness and paralysis of the limbs due to hypocalcemia (Clinigene Co., Ltd., "Foscavir (registered trademark)/Interview Form 2019 March Edition", 2019).

(Reference Example 7) Oral absorption of foscarnet

(7-1) Oral absorbability in laboratory animals

After oral administration of ¹⁴ C-foscarnet dissolved in sterile water to mice (24 mg/kg), rats (24, 96 mg/kg), and dogs (60 mg/kg), the absorption rate of any one of them was approximately 50%, T _max is 15 minutes (mouse), 30 minutes (rat), 1.4 hours (dog), T _{1/2 is} about 8 hours (mouse, rat), 33 hours (dog). (Clinigene Co., Ltd., "Foscavir (registered trademark)/Interview Form March 2019 Edition", 2019)

(7-2) Oral absorption in humans

In 6 patients with AIDS complicated by CMV omentitis, the absorption rate (measured via urine excretion) when 4 g of foscarnet was administered orally at 6 hour intervals and 3 days orally was as follows (Table 9: Source : CIV, Non-Patent Document 15) shows the method is about 18%.

(Example 1) The effect of intracellular uptake of amyloid β (including oligomers) in human-derived nerve cells infected by viruses (HHV-6, HHV-7) inhibited by foscarnet

In this experiment, the study aimed at the uptake of amyloid β (Aβ) in virus (HHV-6, HHV-7) infected cells, and the inhibitory effect of foscarnet on the uptake in cells. The sequence of this test is based on the following method.

(1-1) Preparation of virus solutions for HHV-6A, HHV-6B and HHV-7

According to the following test materials and procedures, make each of the above virus solutions used in the test

(a) Test materials

1) The following cell lines used in the experiment were accepted by the HHV-6 Foundation (USA/Santa Barbara, California).

˙HSB-2 strains: HSB-2 cells not infected, the main species, 1.0mL, @ 1 × 10 ⁷ cells, 12/17/2018, ( BIOCELL Diagnostics H10-865); using 10% FBS, 1 × ATB added IMDM culture medium (Reference: CCRF-HSB-2(ATCC CCL-120.1)Product Sheet)

˙S _u p-T1 strain: Sup-T1 uninfected cells, the main species, 1.0mL, @ 1 × 10 ⁷ cells, 12/17/2018, ( BIOCELL Diagnostics H10-864); using 10% FBS, 1 × ATB supplemented RPMI-1640 culture medium (reference: Sup-T1 (ATCC CRL-1942) Product Sheet)

˙HHV-6A/HSB-2 strain: main species, HHV-6A, GS strain, infected HSB-2 cells, 1.0mL, @5×10 ⁶ cells/mL, 8/13/2018, (BIOCELL Diagnostics H10 -849); cultured in the same medium as HSB-2 (10% FBS, IMDM medium supplemented with 1×ATB)

˙HHV-6B/Sup-T1 strain: Sup-T1 main species, HHV-6B, Z29 strain, infected Sup-T1 cells, 1.0mL, @5×10 ⁶ cells/mL, 8/16/2018 (BIOCELL Diagnostics H10-850); cultured with the same medium as Sup-T1 (10% FBS, 1×ATB supplemented RPMI-1640 medium)

˙HHV-7/Sup-T1 strain: main species, HHV-7, JI strain, P1, infected Sup-T1 cells, 1.0mL, @5×10 ⁶ cells/mL, early passage, 12/2/ 2018 (BIOCELL Diagnostics H10-859); cultured in the same medium as Sup-T1 (10% FBS, 1×ATB supplemented RPMI-1640 medium).

2) FBS: Fetal Bovine Serum, qualified, Brazil (lot 42Q6170K: Thermo Fisher Scientific 10270)

3) ABT: Antibiotic/antifungal mixed solution (lot L7P3293: Nacalai tesque 09366-44)

4) IMDA medium: IMDA, GlutaMAX Supplement (lot 2003869: Thermo Fisher Scientific 31980-030)

5) RPMI-1640 medium: RPMI-1640 medium, GlutaMAX Supplement (lot 1967676: Thermo Fisher Scientific 61870-036)

(b) Cultivation of non-infected cells

In order to obtain the virus solution, the non-infected HSB-2 strain of HHV-A, HHV-6B, and the Sup-T1 strain of HHV-7 were cultured in the following order.

1) The tube for freezing and preserving cells is quickly thawed in a warm bath at 37°C. For HSB-2 cells, mix with 10% FBS and 1×ATB-added IMDM medium, and for Sup-T1 cells with 10% FBS and @1×ATB-added RPMI-1640 medium (9 mL each) was mixed.

2) The above-mentioned mixed liquid was centrifuged (300×g, 5min) with a centrifugal evaporator (S/N 11600775; EYELA CVE-2200), and the supernatant was discarded.

3) After the supernatant was discarded, the cell pellets were scattered by flicking, and each was suspended in a new medium.

4) HSB-2 and Sup-T1 were sown in a suspension culture vessel at a cell density of @2~3×10 ⁵ cells/mL and cultured at 37°C and 5% CO ₂ . Proliferate to 1~2×10 ⁶ cells/L on one side, and dilute 3 to 5 times with new medium for subculture.

(c) Cultivation of virus-infected cells

According to the "Procedure for Subsequent High-potency Viruses, http://hhv6fiundatio.org (RESEARCH-Lab). Proposal: 2019.02.08) set by HHV-6 in the United States, culture cells infected with each test virus.

1) About HHV-6A/HSB-2 (HSB-2 strain infected with HHV-6A) and HHV-6B/Sup-T1 (Sup-T1 strain infected with HHV-6B) and HHV-7/Sup-T1 (Sup-T1 strain infected with HHV-7), freeze-preserved cells (5×10 ⁶ cells each) were quickly thawed in a warm bath at 37°C.

2) The same amount (5×10 ⁶ cells) of non-infected cells (HSB-2 for HHV-6A/HSB-2, Sup-T1 for HHV-6B/Sup-T1 and HHV-7/Sup-T1) , The same below) mix the total volume to 5mL, and dispense into 2 T-75 flasks (2.5mL/flask).

3) Incubate each flask for 2 hours (37°C, 5% CO ₂ ) with stable stirring in an inclined state.

4) Add 10 mL of new medium to each flask.

5) Incubate at 37°C and 5% CO ₂ .

(d) Calculation of virus infection rate

Regarding the virus infection rate of infected cells, the TC20 automatic cell counter (S/N 508BR1149; Bio-Rad) is used to regularly determine the number of cells stained with trypan blue (the number of whole cells, the number of live cells; cell size range: 7~ 36μm, 12~36μm[>12μm]). Compared with the measurement results of non-infected cells (HSB-2 or Sup-T1), the infection rate was calculated using the following formula.

The number of infected live cells = the number of live cells of infected cells × (the ratio of the number of live cells> 12 μm of infected cells-the ratio of the number of live cells> 12 μm of non-infected cells)

Infected cell specific dead cell number = total number of infected cells × (survival rate of non-infected cells-survival rate of infected cells)

Infection rate (%) = (number of infected cells + number of specific dead cells of infected cells) / total number of infected cells × 100

(e) Preparation of virus solution

(e-1) Sequence

HSB-2 or Sup-T1 (equivalent to 5 times the amount) is added to the appropriate amount of new medium at the same time, and the culture is continued until the infection rate becomes about 60-80%. After that, the cells and the culture medium were collected in a 50 mL centrifuge tube at the same time, and frozen and stored (-80°C). After that, the virus solution was prepared in the following procedure.

1) Defrost rapidly in a warm bath at 37°C, and mix with a vortex mixer for 30 seconds. After freezing and storing, quickly thaw and mix again in the same manner with a vortex mixer.

2) Centrifugal separation (1,500 rpm, 10 min, 4°C), and recover the supernatant (SN1) and precipitation (PT1).

3) SN1 is centrifuged in the same manner, and the supernatant (SN2) and precipitation (PT2) are collected.

4) Suspend PT1 and PT2 in separate 1 mL medium, and mix them together (PT3).

5) After PT3 is rapidly frozen with dry ice/ethanol, it is rapidly thawed in a warm bath at 37°C, and this operation is performed twice in total.

6) Centrifugal separation with PT3 (1,500 rpm, 10 min, 4°C), and recover the supernatant (SN3).

7) After mixing SN2 and SN3, centrifugal separation (4,500 rpm, 20 min, 4°C), and recover the supernatant (referred to as SN4).

8) SN4 is filtered with a 0.45μm filter and then concentrated with a centrifugal ultrafilter. Dispense 200~300μL each and freeze and store (-80℃: virus solution)

(e-2) Determination of virus GC in virus solution

Regarding 50 μL of each virus solution obtained above, the number of virus genes (GC) was measured in the following procedure.

1) According to the sequence of DNeasy blood and tissue kit (lot 151046692: QIAGEN 69504) for purification of total DNA from animal blood or cells (spinning column protocol), perform sample adjustment immediately before adding the column containing RNase A treatment , Use Monarch DNA Cleanup Columns to adjust DNA according to the sequence of Monarch PCR & DNA Cleanup Kit (lot 0071711: NEB T1030L).

2) The DNA extracted from 1 μL of each virus solution was used as a template, and the reaction conditions were carried out at a total of 8 μL of 0.3 μM primers (shown in the table below), 1×SsoAdvanced Universal SYBR Green Supermix (lot 64098857: Bio-Rad 1725271) Real-time PCR analysis. CFX Connect Real-Time PCR Detection System (S/N 788BR04674: Bio-Rad) was used as the measurement device.

3) Simultaneously carry out HHV-6 PCR products with a known concentration (HHV-6B is used as a template, and the HHV6-orf67-F1 primers and HHV6-orf67-R1 primers are PCR-amplified products through the column purification), or HHV-7 PCR products (Using HHV-7 as a template, using Hs-ACTB-gF1 primers and Hs-ACTB-gR1 primers to produce PCR-amplified products through the column purification) serial dilutions, real-time PCR analysis, and making a calibration line ( The relationship between C ₁ valence and the number of template DNA (number of viral genome sets: GC)). Based on this calibration curve, the GC in each virus solution was calculated. The results are shown in the following (Table 11).

(e-3) Determination of power price

The determination of the potency of viruses in infected cells is carried out in the following manner.

1) Uninfected HSB-2 (HHV-6A subjects) or Sup-T1 (HHV-6B and HHV-7 subjects) were prepared to 3.3×10 ⁵ cells/mL, and 0.45 mL/well was sown into 24-well plates. Train overnight.

2) 50μL of each virus solution was diluted ²⁰ ~ medium ^2-5 modulation diluted solution.

3) Add 50 μL of the stage-diluted virus solution to uninfected HSB-2 or Sup-T1 (final cell density is 3×10 ⁵ cells/mL), and culture for 2 days.

4) Calculate the calculated infection rate through the sequence specified in the aforementioned "Aβ ₄₂ addition and virus infection" to determine the viral power (IFU/mL).

(e-4) Determination of the number of virus genome sets (GC) in the virus solution

The number of virus genome sets (GC) in each virus solution obtained through the above procedure was determined according to the following procedure.

1) Adjust genomic DNA with 50 μL of virus solution. According to the sequence of DNeasy blood and tissue set [purification of total DNA from animal blood or cells (spin-tube solution)], perform sample adjustment immediately before adding the column containing RNase A treatment.

2) Use Monarch DNA Cleanup Column to purify DNA according to the sequence of Monarch PCR & DNA Cleanup Kit (lot 0071711; NEB T1030L).

3) The DNA extracted from 1 μL of the virus solution was used as a template, and Real-time PCR analysis was performed under the reaction conditions of 0.3 μM primers and 1×SsoAdvanced Universal SYBR Green Supermix in a total of 8 μL.

4) Use CFX Connect Real-Time PCR Detection System (S/N 788BR04674; Bio-Rad) as the measurement machine.

(1-2) Modulation of Aβ ₄₂

In order to test whether the extracellular Aβ is taken into the cell, or whether it is related to HHV-6A, HHV-6B, and HHV-7 viral infections, the test Aβ _{42 was} adjusted in the following order.

1) Aβ ₄₂ powder (starch-like β-protein (Human, 1-42), 0.59 mg; MW 4514.0; lot 680405, PEPTIDE Research Institute, 4349-v) was allowed to stand at room temperature for 30 minutes.

2) Measure HFIP (1,1,1,3,3,3-hexafluoro-2-propanol; lot EKWML-ON; TCI H02424) about 131μL in a syringe barrel connected to the injection needle in the exhaust cabinet. After adding the rubber stopper into the Aβ ₄₂ powder container, mix (1 mM solution). When the rubber plug is pierced into the injection needle, the vacuum state inside the container is opened.

3) Let stand at room temperature for 0.5~2 hours (singulation).

4) Dispensing the monomer mixture into two low protein adsorption microtubes (1.5mL; Watson PK-15C-500) (66μL/piece), using a centrifugal evaporator (S/N 11600775; EYELA CVE-2200) dry. Freeze storage (dry peptide: -80°C).

5) Return the above-mentioned dried peptide to room temperature, add 13.1 μL of DMSO (Dimethyl Sulfide: lot LKF233.Fujifilm Wako 037-24053), and then apply it to a long sonic breaker (Bioruptor UCD-250, S/N 250581, SONIC/ BIO Co., Ltd.), the lysed cells were disrupted by ultrasonic treatment with a cycle of 160W, 10sec On/10sec Off/30 for 10 minutes.

6) Each 2μL of the above container solution was dispensed into 5 microtubes and then stored frozen (5nM solution: -80℃)

7) After mixing 2mL of 5mM solution with PBS(-) or 98μL of culture medium to adjust to 100μM solution, mix it in a vortex mixer for 30 seconds and let it stand at 4°C for 12-24 hours (oligomerization).

(1-3) Setting of test sample

As a sample used in the test, the settings are as follows. Aβ ₄₂ is 0.1~1,000nM (two-stage dilution with a common ratio of 10) and 6 conditions without additives, Foscavir (registered trademark) is a final concentration of 800μM and 2 conditions without additives, virus solution is HHV-7 alone, HHV-7 Mix with HHV-6A, mix with HHV-7 and HHV-6B, no addition, each with MOI 0.1, 1.0 8 conditions, set the sample treatment group as the following table (Table 12).

(1-4) Preparation of test cells

SH-SY5Y (lot: 17C025, P11, 19April 2017; EACC 94030304) is sown at a cell density of 0.1~10 ⁴ cells/cm ² and cultured at 37°C and 5% CO ₂ . The cells were proliferated until they became confluent, and the obtained cells were suspended and subcultured with TrypLE Express (lot 1869186; thermo fisher Scientific 12604021). The 96-well plate was sown at 1.28×10 ⁴ cells/50μL/well (4×10 ⁴ cells/cm ² ), and cultured overnight in "Ham's F12" medium supplemented with 10% FBS, 1×NEAA, and 1×ATB.

(1-5) Addition of Aβ ₄₂ and virus infection

In the following procedure, the addition of Aβ ₄₂ and the virus infection of the test cells are performed in the culture medium of the test cells.

1) Prepare 25 μL of a 800 μM mixed solution of "Foscavir (registered trademark)" (FCN, Foscavir (registered trademark) for intravenous injection, 24 mg/mL, production number XX26, containing 6 g of foscarnet as an active ingredient).

2) Prepare 25 μL of the virus solution prepared by the aforementioned method.

3) Each tested sample was cultured with Ham's F12 medium (Ham's F-12 with L-glutamine and phenol red, lot TWH7034, Fujifilm Wako 087-08335) supplemented with 10% FBS, 1×NEAA, and 1×ATB for 24 hours And 48 hours.

4) After 24 hours, about 1/2 of the test sample, measure the absorbance at a wavelength of 450 nm (Planck measurement). Add 10 μL of CCK-8 (Cell Counting Kit-8, lot NT161, Dojindo 343-07623), and measure the absorbance at 450 nm after 1 to 4 hours. The value obtained by subtracting the Planck measurement value from the measurement value (Planck subtraction value) was evaluated as the number of cells.

5) After 48 hours, with respect to the remaining 1/2 of the test sample, the number of cells can be measured sequentially in 4).

6) The cells after the absorbance measurement were washed with PBS(-) and frozen and stored (-80°C). With MOI 1.0, only the Aβ ₄₂ measurement under the condition of 48 hours was performed.

(1-6) Determination of Aβ ₄₂

The Aβ ₄₂ in each test sample was measured in accordance with the procedure of "Human β Amyloid (1-42) ELISA kit Woko, High Sensitive" (lot CAR2204, Fujifilm Wako 296-64401).

1) Add 100 μL of aspiration buffer to the cells and mix. Let stand on ice for 15 minutes.

2) Centrifugation (3,150×g, 20min), and recover the supernatant.

3) 50 μL of the supernatant is mixed with 50 μL of the standard diluent attached to the kit (2 times dilution).

4) In the antibody (BAN50) immobilized microwell plate attached to the kit, dilute the standard solution attached to the kit in series (0.1, 0.5, 1, 2, 5, 10, 20pmol/L), 100μL/well of the sample with multiple dilutions and the standard diluent (blank) included in the kit.

5) Seal with the disc seal attached to the kit, and react overnight in refrigeration (4°C).

6) Remove the liquid in the well, and wash 4 times with the cleaning solution included in the 1× set of 300 μL/well.

7) Add 100 μL/well of the HRP-labeled antibody (BC05) solution attached to the kit, and react under refrigeration for 1 hour.

8) Wash 5 times in the same way as above.

9) Add 100 μL/well of the TMB solution included in the kit, and react at room temperature for 30 minutes (shading).

10) Add 100 μL/well of the stop solution included in the kit.

11) Measure the absorbance at 450 nm (within 30 minutes). The Planck measurement value is subtracted from each measurement value to obtain the Planck subtraction value.

(1-7) Test results

Through the above test, the conditions of this test were confirmed as follows.

1) Although Aβ ₄₂ outside the cell is taken into the cell, its concentration is not high. (Picture 8)

2) When there is a test virus outside the cell, the concentration of Aβ ₄₂ in the cell tends to decrease (Figure 8).

These results indicate that the extracellular virus system binds to Aβ ₄₂ and inhibits the uptake into the cell. Aβ ₄₂ is a natural immune response product in the brain and has the ability to capture pathogens such as viruses that have invaded or reactivated in the brain. The effect of is shown to be consistent with the findings.

(Example 2) Confirmation of the effect on "virus/Aβ complex" via foscarnet

(2-1) Intracellular uptake inhibitory effect of "virus/Aβ complex" via foscarnet

Use the aforementioned test sample to measure the concentration of Aβ _{42 in the} test cell and the number of viral DNA to verify the uptake of the "virus/Aβ ₄₂ complex" in the test cell and "Foscavir (registered trademark)" ( Whether foscarnet's intravenous preparation) has the effect of inhibiting the intake.

Regarding the SH-SY5Y cell line used in the test, Aβ ₄₂ and each virus solution were added for 48 hours and then digested, and Real-time PCR was performed to determine the virus GC. The results (Figure 9) are as follows.

1) For the test cells, in the genetic examination, although HHV-6A, HHV-6B and HHV-7 were infected alone, the combined infection of HHV-6A and HHV-7, or HHV-6B and HHV-7 was also observed.

2) It is believed that HHV-6A and HHV-7 are compatible with infection observed in high concentration GC. In addition, in the presence of HHV-7, the GC of HHV-6B was significantly lower. This background means that the infectivity to HHV-6B test cells is low.

3) In the group to which foscarnet was added to the culture medium, the amount of Aβ ₄₂ increased and the GC of HHV-6 tended to decrease. However, this tendency was not observed in the foscarnet non-additive group. This result means that even in the presence of Aβ _42, foscarnet has antiviral effect against the HHV-6.

The results of the test showed that the addition of Foscavir (registered trademark) showed the effect of reducing the number of viral DNA in the test cell, which was achieved through the antiviral effect of Foscavir (registered trademark) or through the "virus/Aβ complex" The inhibition of uptake in cells cannot be confirmed under the test conditions.

Furthermore, in the Foscavir (registered trademark) added population, the DNA number of HHV-6 in the cell decreased with the increase of Aβ. This result shows that the extracellular Aβ ₄₂ line is taken up into the test cell together with the virus under the test conditions, and the Aβ ₄₂ line inhibits the intracellular involvement of the virus, and the addition of Foscavir (registered trademark) does not hinder the inhibition.

The above data supports the evidence to capture the natural immune effect of Aβ in viruses and other pathogens that appear outside the cell (W Eimer et al., Neuron 99(1): 56-63, 2018, RD Moir et al., Alzheimer's & Dementias 14( 2018): 1602-1614,201) is also applicable to HHV-6/7 infection.

(2-2) The effect of foscarnet on the cell dysfunction of HHV-6/HHV-7 in the presence of Aβ

In order to verify the cell dysfunction of HHV-6A, HHV6-B, and HHV-7 in the presence of Aβ, as test cells, each virus solution was added to the neuroblastoma/SH-SY5Y strain to infect the virus. The number of cells after 245 hours and 48 hours. Furthermore, we will study how the addition of Foscavir (registered trademark) affects the number of cells.

Cells were found to modulate Aβ sequence and added with viral infection system ₄₂ described above, and the results of the system as follows.

1) Under the above test conditions, Aβ _{42 is} not harmful to cells. After 48 hours and 24 hours, the cell growth rate increased by more than 30% (Figure 7). However, there was no significant difference between the Aβ _42- added group and the non-added group to the medium.

2) Under the test conditions (800μM), it was shown that Foscavir (registered trademark) inhibited cell proliferation (84% compared with the non-additive population).

3) the number of cells added to each virus population increase, the proposed barrier cell virus Aβ ₄₂ low.

A [beta] at 4) the presence of viruses and ₄₂ non-addition group A [beta] ₄₂ added group, Aβ ₄₂ was added to a constellation of cell number increase. However, in the 1000 nM-added population of Aβ ₄₂ , the number of cells maintained a tendency to decrease compared to when measured 24 hours later.

The above results showed that the cytotoxicity of Aβ ₄₂ was not observed under the test conditions. It is nothing more than cell proliferation even in the presence of virus. Therefore, it shows that Aβ ₄₂ has the effect of protecting cells from viruses, which can also be confirmed in HHV-6/7. .

(2-3) Antiviral effect of foscarnet in the presence of Aβ

There are several prior literatures attempting to study the antiviral effect of foscarnet. However, there is currently no experiment to review the effect of foscarnet on HHV-6 and HHV-7 in the presence of amyloid β.

In this article, the antiviral effect of foscarnet against HHV-6A, HHV-6B, and HHV-7 in the presence of Aβ ₄₂ is to use neuroblast cell SH-SY5Y strain to perform Real-time PCR analysis of viral DNA in cells (GC) method verification.

As a result, in the population to which the test solution (800 μM) of Foscavir (registered trademark) was added, the amount of extracellular Aβ ₄₂ increased, and the number of intracellular gene bodies of HHV-6 decreased. However, in the non-added group of Foscavir (registered trademark), there is no tendency to decrease by these methods, showing the antiviral effect of Foscavir (registered trademark) in the presence of Aβ ₄₂ against HHV-6/7 (Figure 9) ). In other words, as a result of the antiviral effect of Foscavir (registered trademark), the virus or infected cells in the cell are reduced. This reduction of intracellular virus can be expected to suppress cell death accompanying virus infection.

(Example 3) In the presence of amyloid β (including oligomers), the inhibitory effect of foscarnet on the calcium uptake of nerve cells

After culturing SH-SY5Y cells on 96-well microplates, add only Aβ, and add Aβ and foscarnet to the culture medium for further culture for 1 to 3 days. After removing the culture medium so as not to damage the cells and washing, add a fluorescent substrate, and measure the amount of calcium in the cells through fluorescence intensity measurement.

Considering the results of the aforementioned (Reference Example 6), in the cells to which foscarnet was added, the uptake of Aβ and/or Aβ oligomers inhibited the increase of intracellular calcium concentration, and it was concluded that the intracellular calcium concentration was kept constant .

(Example 4) Anti-β secretase effect of foscarnet

Since foscarnet has anti-metal (zinc) enzyme inhibitory activity, whether it has the effect of blocking the β-secretase activity of aspartic acid protease (with zinc in the active site) is verified by the following test.

1) As the test material, Foscavir (registered trademark) (Foscavir (registered trademark) for intravenous drip injection 24mg/mL, production number XX26. Contains foscarnet 6g as the main ingredient), "SensoLyte 520 BASE1 Assay Kit made by ANASPEC" Fluorimetric ^TM "(model AS-71144), the positive control inhibitor /LY2886721 attached to the same set, the commercially available CEM company inhibitor ADZ3839 (free base) (CS-5933).

2) Test method

Regarding Foscavir (registered trademark), a 1 mM test solution was adjusted by 190 times dilution of 0.19 M with the buffer supplied with the kit to prepare 5 specimens with final concentrations of 100 μM, 10 μM, 1 μM, 10 nM, and 1 nM.

The inhibitor/LY2886721 attached to the kit was prepared at 250 nM, and the commercially available inhibitor AZD3839 was a sample of 10 μM, and the test was performed according to the test protocol of the kit.

3) Results

For any specimen of Foscavir (registered trademark), in the test conditions, almost no β-secretase blocking effect was seen (Figure 6). This result shows that foscarnet is not Aβ in the treatment of AD, but the invaded HHV-6/7 or reactivated latent HHV-6/7 in the brain is used as the direct treatment target, and the result has a decrease As a function of the formation of Aβ formed by the autoimmune response to viral pathogens.

(Example 5) The killing effect/effect of HHV-6/7 against infected cells

To non-infected cells (HSB-2 for HHV-6A, Sup-T1 for HHV-6B and HHV-7), add the virus solution obtained through the above procedures, and observe the presence or absence of virus infection and changes in the morphology of infected cells by the following methods .

(5-1) About HHV-6A

Add the HHV-6A virus solution obtained by the above procedure to the medium for culturing only HSB-2 (uninfected strain) and the medium for cultivating uninfected strains, take samples from each medium, and observe each medium with an optical microscope (Olympus CKX 41) Cell morphology.

The result is shown in Figure 10-1 (uninfected cells). The shape or size of each cell is uniform, and almost no dead cells are observed. However, in Figure 10-2 (with HHV-6A added), hypertrophy and dead cells are scattered.

(5-2) About HHV-6B and HHV-7

Add the HHV-6B virus solution or HHV-7 virus solution obtained by the above procedure to the medium for culturing only SupT-1 (uninfected strain) and the medium for cultivating the uninfected strain. The sampled medium is subjected to optical microscope (Olympus CKX 41) Observe the cell morphology in each culture medium at 20 times, and take photos with Leica MC 120HD automatic eye mode and HD 1080-50 resolution.

As a result, in Figure 11-1 (cells not infected with virus), no dead cells were seen, and the shape and size of the cells were uniform. However, in Figure 11-2 (with HHV-B added) and Figure 12 (with HHV-7 added), in addition to dead cells, the cell surface is scattered with rough cells and hypertrophic cells.

(5-3) Confirmation of virus infection of test cells by immunostaining method

When the uninfected HSB-2 and Sup-T1 in the culture medium are added with the respective virus solutions obtained through the aforementioned procedures, as to whether the uninfected cells are infected with the virus, the immunostaining test is performed in the following order using antibodies against the proteins that constitute HHV, Inspection certificate.

1) HSB-2 and Sup-T1 were seeded on 24-well plates at 7.6×10 ⁴ cells/0.25 mL/well (3.3×10 ⁵ cells/mL). Train overnight.

2) The virus solutions of HHV-6A, HHV-6B, and HHV-7 obtained through the above-mentioned procedure are added so as to become MOI 1.0. Cultivate for 48 hours.

3) Transfer the entire amount of medium containing each cell to a 1.5 mL tube.

4) Remove the virus-containing medium and wash with PBS(-) (lot.TWK7042, Fujifilm Wako 166-23556) twice.

5) Add ice-cold acetone to each cell and keep it on ice for 10 minutes.

6) Remove acetone and wash with PSB(-) containing 0.5% BSA for 3 times.

7) Add the primary antibody diluted 200 times with FBS(-) containing 0.5% BSA, and incubate at 4°C overnight. Remove the liquid containing the primary antibody and wash 3 times with PBS(-) containing 0.5% BSA.

The primary antibodies used for immunostaining in this experiment are as described below (Table 13).

8) Add a 5-fold diluted secondary antibody (TaKaRa POD conjugated anti-mouse, for tissue, lot AE 45500, Takara MK 204) to each cell, and incubate at room temperature for 1 hour.

9) Wash with FBS(-) containing 0.5% BSA 3 times.

10) Add DAB coloring solution (TaKaRa DAB substrate, lot AI2P021, Takara MK 210), and develop color at room temperature for 5 minutes.

11) Remove DAB hair coloring solution from each cell, wash with PBS(-), and inspect under microscope (Olympus CKX 41)

In uninfected HSB-2 and Sup-T1 cells without the addition of HHV protein (each virus solution), no positive reaction due to antibodies (dark brown staining due to DAB) was seen (Figure 13-1, Figure Figure 13-2, Figure 13-3). On the other hand, in the cell population to which the virus solution was added, positive reactions due to antibodies were seen in most of the cells (Figure 13-4, Figure 13-5, Figure 13-6).

In this way, the viruses (HHV-6A, HHV-6B, and HHV-7) used in the infection test for each uninfected cell line used in the test were confirmed. Furthermore, the stained part showing infected cells is expanded to cells showing normal morphology, which means that living cells exist even under virus infection. Moreover, each cell line used in the test showed a high infection rate against the test virus.

(5-4) Test results

According to the above-mentioned experiments, in the cells infected with HHV-6A, HHV-6B, and HHV-7, there are some irregularities on the cell surface, which is called hypertrophy. Among them, the cells that produce bumps on the cell surface have no change in particle size from non-infected cells. Because of apoptosis, changes in the morphology of the cell membrane are used as the starting point, and are shown as apoptotic cells.

On the other hand, since the hypertrophy of the cell becomes a typical form of necrosis (Reference 15), the hypertroped cell is considered to be a necrotic cell. Furthermore, although there are many cells showing a positive reaction in immunostaining, since the number of cells that become apoptosis and necrosis is small, it is known that most of the infected cells show a normal state (morphology).

Up to now, although virus-infected cells are considered to be apoptotic (Non-Patent Documents 10 and 11, References 30, 31, 37, and 38), through the above test, it is the first time that HHV-6A, HHV-6B, and HHV -7 Infected cells not only apoptosis, but also cause necrosis.

The above findings indicate that HHV-6A or HHV-6B which is latently infected with nerve cells or glial cells in the brain in AD, especially in the hippocampal gyrus where neuronal cell renewal is carried out, contains apoptotic cells. Reactivation at the time of death means that it also induces necrosis of T cells that migrate to the site of inflammation, and various inflammation-causing substances (such as HMGB-1) that are DAMPs (damage/harm related molecular forms) are released from the cells. , TDP-43, GSK-3β, glutamic acid, Ca ^++, etc.) increase inflammation and cause various pathologies that accompany it, especially the excessive death of nerve cells (Non-Patent Document 18 and Q Chen et al., Signal Transduction and Targeted Therapy, 3(18): 1-11, 2018).

Claims (11)

A medicinal composition, which is a medicinal composition for the treatment or prevention of Alzheimer’s disease ("AD"), for human herpesvirus type 6 A and type 6 B (hereinafter, collectively referred to as "HHV-6" ) And Type 7 ("HHV-7") compounds with antiviral activity as active ingredients and contain pharmaceutically acceptable carriers. The pharmaceutical composition according to the first item of the patent application, wherein the aforementioned compound is a compound that further has antiviral activity against herpes simplex virus type 1 ("HSV-1") and cytomegalovirus ("CMV"). The pharmaceutical composition according to item 1 or 2 of the scope of patent application, wherein the aforementioned compound is a compound that binds to the pyrophosphate binding site of a DNA polymerase derived from the target virus and selectively inhibits virus proliferation. The pharmaceutical composition according to any one of items 1 to 3 in the scope of the patent application, wherein the aforementioned compound is phosphinyl acetic acid or phosphinyl carboxylic acid represented by the following basic skeleton (Formula 1) or derivatives thereof Pyrophosphate analogues,

The pharmaceutical composition according to any one of items 1 to 4 in the scope of the patent application, wherein the aforementioned compound is the hexahydrate of 3 sodium phosphonoformic acid represented by the following (Formula 2) (hereinafter referred to as "phosphonic acid "), marked as Foscarnet Sodium in the (JNN)/INN table,

The pharmaceutical composition described in item 5 of the scope of patent application is administered in combination with at least one preparation selected from the following group: ganciclovir, valacyclovir, penciclovir, and At least one nucleoside analog selected by the group consisting of revudine; preparations of cidovir, its prodrugs or derivatives or other nucleotide analogs; preparations of nucleoside compounds; non-nucleoside DNA polymerase inhibitory compounds Preparations of Amonavir or other helicase/initiating enzyme blocking compounds; blocking compounds for the packaging of viral DNA capsids; blocking agents for letevivir or other viral DNA terminal enzyme complexes; containing navel Monoclonal antibody or other PD-1 antibody, PD-1 receptor antibody preparation. The pharmaceutical composition described in item 5 of the scope of patent application is administered in combination with at least one preparation selected from the group of: donepezil, galantamine, rivastigmine, or memantine At least one AD therapeutic agent or a regulator for the movement of neurotransmitters selected from the group consisting of amine preparations, neuroprotective agents, and neurotransmitters; drugs targeting amyloid β or Aβ oligomers; Tau protein as Targeted drug; inhibitors of excessive calcium influx to cells; vaccines, ibuprofen, ginkgo extract, or other anti-inflammatory agents; insulin or other anti-diabetics; anti-IL-6 antibodies, anti-IL-12 antibodies, Or other neutralizing agents of inflammatory cytokines and chemokines. A pharmaceutical composition for diagnosing the risk of AD or the progression of AD. It contains DNA or protein derived from HHV-6A, HHV-6B and/or HHV-7, or specific to HHV- 6A, HHV-6B and/or HHV-7 antibodies or fragments thereof, as compounds to be detected as AD biomarkers. The pharmaceutical composition according to item 8 of the scope of the patent application, wherein the aforementioned pharmaceutical composition contains HHV-6A derived from the subject's cerebrospinal fluid (CSF), plasma, blood, or saliva , DNA or protein of HHV-6B and/or HHV-7, or a compound specific for HHV-6A, HHV-6B and/or HHV-7 antibodies or fragments thereof. A pharmaceutical composition for confirming the therapeutic effect of an antiviral agent used in the treatment of AD, comprising a viral DNA or viral protein derived from HHV-6A, HHV-6B and/or HHV-7 , Or compounds specific for HHV-6A, HHV-6B and/or HHV-7 antibodies or fragments thereof as AD biomarkers to be detected. The pharmaceutical composition according to item 10 of the scope of patent application, wherein the aforementioned pharmaceutical composition contains HHV-6A, HHV-6A, HHV-6A, HHV-6A, CSF, plasma, blood, or saliva of the subject. HHV-6B and/or HHV-7 DNA or protein, or a compound specific for HHV-6A, HHV-6B and/or HHV-7 antibody or a fragment thereof.

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