WO2020138402A1 - Antiviral agent for treating or preventing alzheimer's disease and use thereof - Google Patents

Antiviral agent for treating or preventing alzheimer's disease and use thereof Download PDF

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WO2020138402A1
WO2020138402A1 PCT/JP2019/051379 JP2019051379W WO2020138402A1 WO 2020138402 A1 WO2020138402 A1 WO 2020138402A1 JP 2019051379 W JP2019051379 W JP 2019051379W WO 2020138402 A1 WO2020138402 A1 WO 2020138402A1
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hhv
cells
virus
foscarnet
pharmaceutical composition
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Japanese (ja)
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一博 川端
良和 中村
孝明 柳
千登勢 折居
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クリニジェン株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a therapeutic agent for cranial nerve diseases, particularly Alzheimer's disease and its use.
  • AD Alzheimer's disease
  • ADL instrumental activities of daily living
  • basic ADL basic ADL
  • a ⁇ amyloid ⁇ protein
  • a ⁇ oligomers accumulation of its aggregates
  • IL-6 amyloid ⁇ protein
  • a ⁇ oligomers accumulation of its aggregates
  • IL-6 IL-6
  • IL-12 IL-12
  • chemokines such as CCL2
  • brain parenchymal calcium concentration IL-6, IL-12, TNF- ⁇ , and chemokines
  • a ⁇ is considered to be a key factor for the onset of AD, such as elevation and an abnormality in glucose metabolism, which induces neuronal necrosis (Non-patent Document 1, References 2 and 3).
  • a ⁇ adheres to cerebral blood vessels to cause inflammation and often cause amyloid angiopathy (reference document 3).
  • Amyloid angiopathy in the brain leads to weakening and disruption of BBB (reference document 4) and promotes migration of T cells and NK cells in peripheral blood to inflammatory sites.
  • AD hyperphosphorylation and aggregation of tau protein, which is the main component of microtubules, accompanying neurofibrillary tangles, and neuronal cell death, and in patients with advanced symptoms, brain (especially hippocampus) atrophy and large amounts. Neuronal cell death is observed (Reference 5).
  • the cause of cell death, which can be said to be excessive in AD, is considered to be "apoptosis" as a pathology of A ⁇ (in particular, an oligomer mainly composed of A ⁇ 42) accumulated in cells (References 1 and 5).
  • Non-Patent Document 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” Vol. 57, No. 3, 95-109, 2017
  • Non-Patent Document 3 Non-Patent Document 3
  • DMTs disease-modifying therapies
  • Disease-Modifying Therapies Development of AD drugs based on iPS cells and the like
  • Reference 6 AA Fabregat et al., CNS Drugs (2017) 31: 1057-1082, 2017
  • Non-Patent Documents 4 and 5 DAMPs are a group of substances released from cells that have been injured and died for some reason, and have sources such as the nucleus, cytoplasm, mitochondria, and endoplasmic reticulum, and the substances released are also diverse (Non-Patent Document 5, Reference Reference 7).
  • those related to AD include A ⁇ , calcium (reference document 8), HMGB-1 (reference document 9), TDP-43 (reference document 10), GSK-3 ⁇ (reference document 11), glutamic acid/glutamine.
  • Non-Patent Document 7 This indicates the importance of suppressing the death of neural stem cells or cells in the process of differentiation rather than prolonging the life of nerve cells that have completed maturation and then apoptotic in the treatment of AD.
  • neural stem cells existing in the lower part of the posterior part of the brain are differentiated into neurons and then migrate to the olfactory bulb, but it is unknown whether they become neurons in other tissues of the brain, for example, neocortex of the cerebrum Is.
  • HSV-1 herpes simplex virus type 1
  • AD herpes simplex virus type 1
  • HSVE severe herpesvirus encephalitis
  • HSV-1 is a risk factor for AD development, and it has been pointed out that HSV-1 infection leads to A ⁇ accumulation and excessive phosphorylation of tau protein for more than 10 years (non-patent document 8) The proof that HSV-1 is the main cause of AD has not been obtained yet.
  • 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
  • HHV-6 encephalitis is a disease caused by reactivation of latent virus showing a clinical picture similar to AD, and the main causative virus is human herpesvirus 6B (HHV-6B).
  • HHV-6 encephalitis is HHV-6 (especially HHV-6B) that is latently infected in the brain under the condition of weakened immunity such as initial infection in infancy or transplantation of hematopoietic stem cells, bone marrow, and organs. It develops when the reactivation of erythrocytes is accelerated (mass multiplication of virus) (references 20 to 22).
  • HHV-6 encephalitis is a disease caused by reactivation of latent virus showing a clinical picture similar to AD, and the main causative virus is human herpesvirus 6B (HHV-6B).
  • HHV-6 encephalitis is HHV-6 (especially HHV-6B) that is latently infected in the brain under the condition of weakened immunity
  • Reference 20 A Ansari et al., Emerging Infection Disease 10 (8), 2004 Reference 21: Masao Ogata, "Clinical Blood," Vol. 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: Kyoko Taya et al., National Institute of Infectious Diseases, Pathogen Detection (Detection) Manual, “Examination of Rash", http://www.niid.go.jp/niid/ja/labo-manual.html
  • HHV-6 Human herpesvirus type 6 (HHV-6) and type 7 (HHV-7)
  • HHV-6 is a DNA virus having double-stranded DNA as its genome, which was discovered in 1986 and HHV-7 in 1990, and belongs to the herpesvirus family B subspecies.
  • HHV-6A and HHV-6B are latent infection destinations of CNS tissue (such as brain glial cells and hippocampus/neuron).
  • CNS tissue such as brain glial cells and hippocampus/neuron.
  • HHV-7 can latently or infect salivary glands and olfactory bulb.
  • HHV-6A CD46 (expressed in most cells) as a main receptor of the host cells invaded by the above viruses
  • HHV-6B is CD134 (expressed in CD4 positive and/or CD8 positive T cells) in addition to CD46
  • HHV-7 is known to have CD4 and CD8 (expressed in activated T cells etc.) (references 25 and 26).
  • HHV-6 (A and B), which was not eliminated by the immune system, is latent in host cells, but the method is unique and the viral genome is integrated with the telomere of the human chromosome.
  • one chromosome of the host cell incorporates only one HHV-6 (A or B) genome, and X, 1, 6, 7, 9, 10, 11, 12, 17, 18, as the chromosome into which the viral genome enters. Chromosomes 19 and 22 are known (reference document 28), but latency in other chromosomes has not been confirmed.
  • About 1% of all human beings inherit the HHV-6A or HHV-6B genome by inheritance, but the pathological effects of this virus (iciHHV-6A and iciHHV-6B) are poorly understood.
  • HHV-7 also has the same repeated nucleotide sequence ([TAACCC]n) as HHV-6 (A and B) at both ends of the genome, and is involved in the development of HHV-6 encephalitis-like disease (Reference 29), It has not been clarified whether HHV-6 (A and B) is latently infected with telomeres of human chromosomes. As a disease caused by HHV-7 infection, an exanthema subitum, chronic fatigue syndrome, etc., which are rarely caused after the initial infection of HHV-7 in infancy, are known (reference document 26).
  • HHV-6B is not only HHV-6 encephalitis, but also a major causative virus of idiopathic rash in infants and encephalitis at initial infection (References 25 and 26). Most of HHV-6 that causes clinically observed pathological effects is HHV-6B, but the cause thereof has not been clarified. The pathology when HHV-6A and HHV-7 are reactivated has not been fully elucidated, but HHV-6A leads infected cells to apoptosis similarly to HHV-6B (Non-patent document 10, reference document 30). Moreover, HHV-6A and HHV-7 can be co-infected (reference 31).
  • Non-patent Document 11 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 first symptoms of HHV-6 encephalitis can answer distant past events, but can not remember short-term memory such as daily events (short-term memory disorder), do not know the location of their room or toilet, Such disorientation (cognitive impairment) is similar to the typical symptoms of early AD.
  • consciousness disorder (62%), disorientation (52%), memory disorder (48%), convulsion (32%), sensory disorder (22%), independence.
  • Neuropathy (6.2%) has been reported (Reference 32). It is reported that the frequency of occurrence is 12 to 30 days after organ transplantation (Ref. 33), and if the start of treatment is delayed, there is a high possibility of serious sequelae and fatalities.
  • MRI examination of the head of patients with HHV-6 encephalitis shows bilateral abnormal findings in the limbic region (medial temporal hippocampus, amygdala) (Reference 34). This is also a pathological finding typically observed in AD.
  • HHV-6 (A and B) or HHV-7 infection is a risk factor for AD development or exacerbation, there is no research or report pointing out as a direct cause of AD.
  • AD therapeutic agents have been studied and developed by research institutions such as pharmaceutical companies and universities according to various hypotheses (Non-patent Documents 3, 4 and References 6, 35).
  • HHV-6A and/or HHV-6B have been unknown, and clinical trials targeting the virus in AD patients have been It has never been tried.
  • compounds having an antiviral effect against HHV-6 (A and B) and HHV-7 and pharmaceutical compositions (formulations) thereof such as foscarnet, famciclovir, acyclovir, valacyclovir, ganciclovir and phosphophos, including cidofovir.
  • Anti-viral agents such as nate derivative, anti-CD40 antibody, pyridoquinoxaline, pyrazolopyridine derivative, fluorescent compound, SITH-1 inhibitor, nano-emulsion composition, gremlin derivative, etc. I can't find anything.
  • AD Alzheimer's disease
  • the present inventor has noted that the history of infection with the latent virus, HSV-1, is involved in the development of dementia, and AD of AD by targeting a brain latent virus that does not normally show infectious symptoms. I thought about treatment. That is, although the latent infection of glial cells and nerve cells in the brain is carried out, in such a situation, the latent virus that does not show the inflammatory symptoms (fever, redness, etc.) associated with the infection is targeted, and its reactivation is carried out by antivirus. It is the prevention and treatment of AD onset by suppressing with a drug.
  • antiviral agents for the treatment and prevention of AD.
  • reactivation of latent virus in the brain promotes the production of A ⁇ as an innate immune response, and the pathology (inflammation and cell Death) is deeply involved in the onset and exacerbation of AD.
  • Non-Patent Document 15 It has been reported that latent infection of virus and its reactivation are based on the synergistic action of various viruses (Non-Patent Document 15). The inventor pays attention to this point, in the brain of AD patients, particularly in the hippocampus, HHV-6A, HHV-6B and HHV-7 known to have latent infection in the brain (hereinafter, referred to as "HHV-6/ It is highly likely that the synergistic effect of "7") will worsen or manifest infection symptoms upon reactivation. Considering that A ⁇ production is an innate immune response to the virus that appears in the brain, when HHV-6/7 is reactivated, A ⁇ produced and released by nerve cells attaches to the surface of these viruses and stops moving.
  • a ⁇ accumulation in nerve cells in AD patients involves at least infection associated with reactivation of HHV-6/7 that was latently infected in the brain, and suppresses this reactivation/infection. Then, it becomes possible to prevent or treat the onset of AD (FIG. 1) associated with A ⁇ .
  • the defense power against the virus may be lowered and the reactivated virus may be allowed to grow.
  • the inventor if the reactivation of this latent virus in the brain, especially HHV-6/7 is the root cause of A ⁇ production, the elimination of such latent virus (specifically, when reactivating Inhibition of A ⁇ production and oligomerization, inhibition of A ⁇ production and its oligomerization, accumulation of A ⁇ (oligomer) in nerve cells, and the resulting rapid increase of Ca ++ concentration resulting in various neuronal cell death It was thought that it could be a fundamental therapeutic drug for AD (Fig. 2).
  • HHV-6 A and B
  • HHV-7 HHV-7
  • the following experimental system was constructed using a cultured human nerve cell line infected with HHV-6/7 as a model cell simulating the nerve cell of.
  • the reason why the test system in the animal model was not constructed is that since HHV-6/7 is a human-specific herpesvirus, an appropriate infected animal model cannot be obtained.
  • Example 5 For HHV-6A, uninfected HSB-2 strain (human T lymphocyte blast cell line), and for HHV-6B and HHV-7 uninfected Sup-T1 strain (human T lymphocyte-derived T cell line) ( Each virus solution obtained in Example 1) was added, and the presence or absence of virus infection and changes in the morphology of infected cells were observed (Example 5).
  • HHV-6/7-infected T cells and the like also induce necrosis in addition to apoptosis, and the pathology caused by these two different types of cell death, particularly necrosis, causes Pathology of A ⁇ for activation (selective lesions/cell death of nerve function, inhibition of Ca ++ homeostasis, synapse damage, activation of microglia and astrocyte reaction, increase of insulin resistance, etc.: Reference 3 ) Is a fundamental cause of AD onset.
  • HHV-6 encephalitis cascade hypothesis The hypothesis of being received (“HHV-6 encephalitis cascade hypothesis") is established. Based on the above, the multi-herpesvirus agent, which has an excellent effect on HHV-6 (A and B) and HHV-7, not only stops the progression of AD but also its fundamental treatment in a short period (3 to 6 months). The present invention has been completed by finding out that it can be realized.
  • AD is considered to be developed and exacerbated by the following processes (“HHV-6 encephalitis cascade hypothesis”).
  • HHV-6 encephalitis cascade hypothesis
  • Most humans are infected with HHV-6 (A and B) and HHV-7 at an early age, and the virus, especially HHV-6 (A and B), is infected with CNS tissues (glial cells in the brain, especially astrocytes and It is latent in the hippocampus and olfactory bulb with reproducible nerve cells (JM Reynaud et al., ISRN Virology 2013, vol.2013: 1-11, 2013).
  • CNS tissues glial cells in the brain, especially astrocytes and It is latent in the hippocampus and olfactory bulb with reproducible nerve cells (JM Reynaud et al., ISRN Virology 2013, vol.2013: 1-11, 2013).
  • the virus In human cells, the virus not only infects and latencies immune cells such as monocytes and CD4 and
  • chemokines such as CCL2 produced by infected cells upon reactivation express CD46 (HHV-6A), CD134 (HHV-6B), and CD4 (HHV-7), which are the main receptors of the virus.
  • CD46 HHV-6A
  • CD134 HHV-6B
  • CD4 HHV-7
  • T cells and the like migrate to the site of infection, they are not phagocytosed but warp and cause the growth of the virus.
  • T cells and the like migrate to the site of infection, they are not phagocytosed but warp and cause the growth of the virus.
  • neural stem cells in the dentate gyrus maintain the homeostasis of the number of neurons by differentiating into progenitor cells, granule cells and cone cells.
  • HHV-6 which is a DNA virus latent in hippocampal neural stem cells and progenitor neurons, is likely to be reactivated. .. 5) Reactivation of latent HHV-6 causes mild inflammation (HHV-6 encephalitis) associated with infection in the vicinity. Neurons that detect this viral infection produce and secrete A ⁇ as an innate immune response, which captures the virus or is detected by astrocytes and transmitted to microglia, which plays the role of macrophages in the brain, and processes it. Entrust.
  • T cells are activated and migrate through the BBB to the infected site.
  • inflammatory cytokines such as TNF- ⁇ and IFN and chemokines such as CCL2
  • T cells are activated and migrate through the BBB to the infected site.
  • chemokines such as CCL2
  • a ⁇ plaques senile plaques
  • the BBB that is first vulnerable to aging is the hippocampal BBB (A Montagne et al., Neuron, 85 (2):296-302, 2015).
  • NK cells and activated T cells in peripheral blood pass through the weakened BBB by the attraction of chemokines such as CCL2 and the inflammatory site of the brain To run to.
  • chemokines such as CCL2
  • CCL2 chemokines
  • NK cells which is a major attacker against pathogens such as viruses
  • cell death mostly apoptosis due to NK cells and impaired T cells (expressing CD8) is enhanced.
  • HHV-7 that had been latently infected with the olfactory bulb, etc. is reactivated and migrates to the hippocampus, or is reactivated in the hippocampus, the infected cells TRAIL to the surrounding uninfected nerve cells by co-infection with HHV-6A.
  • HHV-6 encephalitis associated with HHV-6 (A and B) reactivation was found in tissues (hippocampus and limbic system) that have neural stem cells and progenitor cells coupled with the action of A ⁇ on viral infection. Pathology such as is manifested, cell death exceeding regenerative ability progresses slowly and surely, nerve function is gradually impaired, and AD develops.
  • a pharmaceutical composition for treating or preventing Alzheimer's disease which comprises human herpesvirus type 6 A and B (hereinafter collectively referred to as “HHV-6”) and/or type 7 (“ A pharmaceutical composition comprising a compound having antiviral activity against HHV-7”) as an active ingredient and a pharmaceutically acceptable carrier, and use thereof.
  • AD Alzheimer's disease
  • HHV-6 human herpesvirus type 6 A and B
  • type 7 A pharmaceutical composition comprising a compound having antiviral activity against HHV-7" as an active ingredient and a pharmaceutically acceptable carrier, and use thereof.
  • an effective amount of a compound having antiviral activity against “HHV-6” and/or “HHV-7” for treating (including prevention) Alzheimer's disease (“AD”) is a further equivalent effective amount, It is desirable that the compound has a multi-antiviral action that is active against herpes simplex virus type 1 (“HSV-1”) and cytomegalovirus (“CMV”) that cause central nervous system diseases such as encephalitis ..
  • HSV-1 herpes simplex virus type 1
  • CMV cytomegalovirus
  • the pharmaceutical composition of the present invention is not only “HHV-6” and “HHV-7”, but also equivalent or similar effective to HSV-1 and CMV which cause central nervous diseases such as encephalitis.
  • a compound having an active amount, that is, having a multi-antiviral effect is used as an active ingredient.
  • Foscarnet trisodium phosphinate hexahydrate
  • JNN trisodium phosphinate hexahydrate
  • the pharmaceutical composition comprises an injection containing foscarnet as an active ingredient, and the effective amount of foscarnet is 60 to 180 mg/kg/day divided into two or three times a day, and the medullary cavity
  • the pharmaceutical composition according to the above [5] and its use which are pharmaceutical compositions for preemptive therapy by continuous administration for 3 to 10 days by internal administration or intravenous drip infusion, can be exemplified.
  • each virus of HHV-6A, HHV-6B and HHV-7 in cerebrospinal fluid and/or blood is used.
  • an effective dose of foscarnet for example, 60 to 180 mg/kg/day, is divided into 1 or 3 times a day, Administration by intracavitary injection or intravenous drip infusion for 3 to 10 days, preferably 3 to 5 days.
  • the period of preemptive therapy and the dose of foscarnet per day or per day can be appropriately adjusted.
  • the pharmaceutical composition comprises an injectable preparation containing foscarnet as an active ingredient, and an effective dose of foscarnet is 60 to 120 mg/kg/day for the purpose of preventing reactivation of each virus.
  • Injection intramuscular injection or subcutaneous injection, which is a pharmaceutical composition for maintenance therapy after pre-emptive therapy by continuous administration for 2 to 6 months, and the pharmaceutical composition according to the above [5] The use can be illustrated.
  • the effectiveness of foscarnet is 60 to 120 mg/kg/day, administered intravenously, intramuscularly or subcutaneously, preferably intravenous drip for 2 to 6 months, preferably 2 to 3 months.
  • Maintenance therapy in consideration of the decrease and maintenance status of DNA, the recovery status of patient's cognitive function, and to prevent or control the occurrence of serious side effects such as maintenance of tolerability of foscarnet or renal damage. And the dosage of foscarnet per dose or per day can be adjusted appropriately.
  • the pharmaceutical composition comprises an oral agent containing foscarnet as an active ingredient, and together with/or in place of maintenance therapy by injection, an effective amount of foscarnet per day is 2000 mg to 6000 mg.
  • a pharmaceutical composition for maintenance therapy which is characterized in that it is continuously administered for 3 to 6 months in 3 to 5 divided doses, and the pharmaceutical composition and its use according to [5] above. It is illustrated.
  • an oral preparation containing the foscarnet of the present invention as an active ingredient is used as a maintenance therapy, in combination with or in place of the maintenance therapy by injection, an effective amount of foscarnet per day is 2000 mg to 6000 mg. , 3 to 5 times a day, continuously for 3 to 6 months.
  • foscarnet has mucosal stimulant properties, teprenone, sucralfate, sodium azulensulforate, repamipide, polaprezinc, irsogladine maleate, bexanate hydrochloride, sofarcone, when administering its oral preparation, A combination with a drug having a gastric mucosal protective action such as cetraxate hydrochloride, ecabet sodium hydrate or the like or a combination thereof is desirable.
  • a drug having a gastric mucosal protective action such as cetraxate hydrochloride, ecabet sodium hydrate or the like or a combination thereof is desirable.
  • the pharmaceutical composition and its use according to the above [5] which is administered in combination with at least one preparation selected from the group of preparations containing -1 receptor antibody.
  • the pharmaceutical composition for treating (including prevention) AD having foscarnet as an active ingredient of the present invention is chemically or different in action mechanism against viruses that cause central nervous system diseases such as encephalitis. It can be administered in combination with a formulation of a compound having an antiviral effect. At that time, they may be administered simultaneously or separately.
  • Preparations of compounds that can be used in combination include preparations of nucleoside analogs such as ganciclovir, valacyclovir, penciclovir and brivudi, preparations of nucleotide analogs such as cidofovir or its prodrugs, derivatives, preparations of nucleoside compounds, non-nucleoside DNA polymerase inhibitory compounds.
  • a helicase-primase inhibitor compound such as amenamevir, a package inhibitor compound for viral DNA capsid, an inhibitor of viral DNA terminase complex such as letermovir, PD-1 antibody such as nivolumab, PD-1 receptor
  • a helicase-primase inhibitor compound such as amenamevir, a package inhibitor compound for viral DNA capsid, an inhibitor of viral DNA terminase complex such as letermovir, PD-1 antibody such as nivolumab, PD-1 receptor
  • At least one AD therapeutic agent or neurotransmitter migration regulator selected from the group consisting of donepezil, galantamine, rivastigmine, or memantine preparations, neuroprotective agents, and neurotransmitters; targeting amyloid ⁇ or A ⁇ oligomers Drug; drug targeting tau protein; inhibitor of excessive calcium influx into cells; vaccine, ibuprofen, ginkgo extract, or other anti-inflammatory drug; insulin or other antidiabetic drug; anti-IL-6 antibody,
  • the pharmaceutical composition according to the above [5] which is administered in combination with at least one preparation selected from the group consisting of anti-IL-12 antibody, other inflammatory cytokine, and a chemokine neutralizing agent. Things and their use.
  • an injection or an oral preparation for treating (including preventing) AD containing foscarnet as an active ingredient of the present invention has a different action mechanism from foscarnet. It may be administered in combination with the AD therapeutic agent ("AD therapeutic agent").
  • agents that target amyloid ⁇ include oligomers
  • tau protein In addition to preparations of donepezil, galantamine, rivastigmine, memantine, neuroprotective agents, neurotransmitters and their regulators, agents that target amyloid ⁇ (including oligomers), and tau protein are targeted Drugs, inhibitors of excessive calcium influx into cells, vaccines, anti-inflammatory agents such as ibuprofen and ginkgo extract, anti-diabetic agents such as insulin, inflammatory properties such as anti-IL-6 antibody and anti-IL-12 antibody There are, but are not limited to, cytokines, chemokines neutralizing agents.
  • a diagnostic agent for diagnosing a person at risk of suffering from AD or the progress of AD, or for confirming the therapeutic effect of an antiviral agent used for the treatment of AD which is in cerebrospinal fluid, plasma or There is a possibility that it can be used for a diagnostic agent containing viral DNAs specific for HHV-6A and HHV-6B in blood or saliva and HHV-7 as biomarkers and their use.
  • ADVANTAGE OF THE INVENTION According to the present invention, AD is initially infected with "HHV-6" and "HHV-7" in infancy, and some people elicit a mild inflammatory reaction (HHV-6 encephalitis, wisdom fever), but at an early stage. Healed and time passed without any major mistake.
  • AD HHV-6 encephalitis-like disease in which "HHV-6" latently infected to hippocampus and limbic cells after the initial infection develops due to reactivation renewed due to a decrease in immunity with age.
  • AD in the cerebrospinal fluid or blood, preferably in plasma, more preferably by using the viral DNA specific for HHV-6A and HHV-6B and HHV-7 in saliva as biomarkers, AD It is possible to confirm the therapeutic effect of an antiviral agent used for diagnosing the progress of AD and treating AD, for persons at risk of suffering from AD.
  • a biomarker and a diagnostic method for AD such as amyloid PET, tau PET, and MRI together, a more reliable diagnosis becomes possible.
  • the first lesions in AD are the hippocampus and its surrounding nerve tissue, which are responsible for the regeneration and division of nerve cells and are responsible for early memory and cognitive functions.
  • the hippocampus and its surrounding nerve tissue which are responsible for the regeneration and division of nerve cells and are responsible for early memory and cognitive functions.
  • the present invention is not a symptomatic drug that only relieves the symptoms of AD (particularly memory/cognitive impairment), but reactivation of "HHV-6" and "HHV-7" that is one of the underlying causes of AD development.
  • the treatment or prevention of AD can be expected to be prevented by preventing the change. From the above, it is possible to suppress an increase in medical expenses for the treatment of AD, and it is possible to significantly reduce the social burden and loss associated with leave and nursing care due to AD.
  • FIG. 2 is a graph showing changes in plasma and cerebrospinal fluid concentrations after oral administration of an aqueous solution containing 4000 mg of foscarnet to 6 AID patients 3 times a day for 3 days.
  • the concentration in plasma reaches a peak immediately after the administration, and decreases to about 1/3 of the peak after 6 hours and to about 1/10 after 12 hours. However, in cerebrospinal fluid, it reached a peak (about 25% of plasma concentration) about 1 hour after administration, and then decreased slowly, and after 6 hours, maintained at 80% or more of plasma concentration for 12 hours. Later it exceeds the concentration in plasma. Even for 12 hours, HHV-6 (A and B) and HHV-7 are kept at a concentration (50 ⁇ M/L) that can ensure an IC 50 .
  • FIG. 5 is a diagram showing the results of a test for examining the effect of HHV-6B on the human-derived T cell line (Sup-T1 strain) in Example 5.
  • the cells were cultured separately in the group to which the virus solution was not added (left figure) and the group to which the HHV-6B virus solution was added (right figure), there was no change in the test cells in the (left figure).
  • each virus solution was added (right panel)
  • a shape unique to apoptotic cells cracked cells
  • a shape unique to necrotic cells swelling, etc.
  • FIG. 5 is a diagram showing the results of a test for examining the effect of HHV-7 on the human-derived T cell line (Sup-T1 strain) in Example 5. Similar to FIGS. 10 and 11, a plurality of cells having a shape unique to the cells undergoing apoptosis or necrosis were observed.
  • FIG. 5 is a diagram showing the results of a test conducted in Example 5 to examine whether HSB-2 strain is infected with HHV-6A by an immunostaining method using an anti-HHV-6A gp82 antibody.
  • FIG. 7 shows the results of a test conducted in Example 5 to examine whether or not HHV-7 is infected with a human-derived T cell line (Sup-T1 strain) by an immunostaining method using an HHV-7KR4 antibody. The presence or absence of infection was confirmed by immunostaining in the HHV-7 virus solution-free group (left figure) and HHV-6B virus-containing group (right figure). However, many cells showed a positive reaction in (right panel). The figure which showed the outline of the AD onset process by "HHV-6 encephalitis cascade hypothesis" that AD develops with reactivation of latently infected HHV-6/7 in the brain.
  • HHV-6 A and B Compounds having an antiviral effect against B) and HHV-7 and pharmaceutical compositions (formulations) thereof are effective.
  • Examples of such compounds and preparations include foscarnet (Japanese Patent Publication No. 10-509704, Japanese Patent Publication No. 11-509515, JP 2007-284452), famciclovir (JP 04-275229), acyclovir. (US Pat. No. 4,957,244), valacyclovir (Patent No.
  • foscarnet and ganciclovir have been confirmed to be effective when applied to HHV-6 encephalitis, and are preferable as active ingredients of a pharmaceutical composition for treating and preventing AD.
  • Particularly preferred is foscarnet, which is known as a multi-antiviral agent.
  • two or more of these compounds may be used in combination and used as a mixture or a combination.
  • Foscarnet When the term “foscarnet” is used in the present invention, it means phosphonoformic acid represented by the following (formula 1) or a salt thereof, or a solvate thereof.
  • the salt is a pharmaceutically acceptable salt, such as lithium salt, sodium salt, potassium salt, magnesium salt, metal salt such as calcium salt, ammonium salt, methylammonium salt, dimethylammonium salt, trimethylammonium salt,
  • ammonium salts such as dicyclohexyl ammonium salt, mineral salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphoric acid, metaphosphate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc.
  • acids valeric acid, stearic acid, oleic acid, gluconic acid, lauric acid, salicylic acid, laocric acid, tannic acid, organic acid salts such as butyl sulfonate, and the like.
  • the solvent capable of forming a solvated acid include methanol, ethanol, isopropanol, acetone, ethyl acetate, methylene chloride, diisopropyl ether and the like.
  • foscarnet sodium hydrate represented by the following (formula 2), and is represented as Foscarnet Sodium in (JNN)/INN notation.
  • JNN Foscarnet Sodium in
  • the compound of the formula (2) will be mainly described as a typical “foscarnet”, but the invention is not limited thereto.
  • Foscarnet is a compound that has a strong antiviral action against HHV-6 and HHV-7 and has an excellent clinical effect on HHV-6 encephalitis that develops after hematopoietic stem cells and organ transplants, and has the following characteristics, and Alzheimer's Most suitable as an antiviral agent for the treatment (including prevention) of diseases.
  • Amyloid ⁇ is produced by degrading amyloid precursor protein (APP) by two amino acid degrading enzymes, ⁇ -secretase and ⁇ -secretase.
  • APP amyloid precursor protein
  • foscarnet has been found to have a low effect of inhibiting the enzyme activity of ⁇ -secretase and/or ⁇ -secretase (Table 4), and an action of suppressing A ⁇ production can be expected.
  • Example 5 For HHV-6A, performed on uninfected HSB-2 strain (human T lymphocyte blast cell line) and HHV-6B and HHV-7 uninfected Sup-T1 strain (human T lymphocyte-derived T cell line) Each virus solution obtained in Example 1 was added, and the presence or absence of virus infection and changes in the morphology of infected cells were observed (Example 5). As a result, in the HHV-6A uninfected cell group (FIG. 10-1), the shape and size of each cell were uniform, and almost no dead cells were observed. However, in the infected cell group (FIG. 10-2), enlarged cells and dead cells were scattered.
  • HHV-6B and HHV-7 dead cells were not found in the uninfected Sup-T1 group (Fig. 11-1), and the cell shape and size were uniform. However, in the HHV-6B-infected cell group (FIG. 11-2) and the HHV-7-infected cell group (FIG. 12), in addition to a few dead cells, rugged cells and swelled cells were scattered. The above test revealed that cells infected with HHV-6A, HHV-6B, and HHV-7 undergo morphological changes such as cell surface unevenness and hypertrophy.
  • cells having irregularities on the cell surface are apoptotic cells because their diameter is the same as that of non-infected cells and in the case of apoptosis, the origin is a change in the cell membrane.
  • hypertrophy of cells is a typical form of necrosis (reference document 24)
  • hypertrophied cells are considered to be necrotic cells.
  • many cells showed a positive reaction by immunostaining, the number of cells that became apoptotic and necrotic was small, which revealed that many of the infected cells showed a normal state (morphology).
  • HHV-6A and HHV-6B which are latently infected with nerve cells and glial cells in the hippocampus in which the neuron is newly formed in AD, undergo cell death including apoptosis of host cells. When reactivated, it also causes necrosis, and various inflammatory substances (eg, HMGB-1, TDP-43, GSK-3 ⁇ , glutamic acid, Ca ++, etc.) from the cells as DAMPs (Damage/Danger Associated Molecular Patterns). Is released, suggesting enhanced inflammation.
  • various inflammatory substances eg, HMGB-1, TDP-43, GSK-3 ⁇ , glutamic acid, Ca ++, etc.
  • Safety Hoscavir registered trademark is a drug with a wide safety margin, with a large difference between the effective dose and the toxic dose for the target HHV-6A, HHV-6B and HHV-7 (Fig. 3). .. Since it has been approved and clinically used in various countries around the world such as Japan, US and Europe for CMV infection (Non-patent Document 13), it can be safely used for treatment with AD.
  • HHV-6/7 is reactivated in the neurons of the hippocampus and its surroundings, which causes A ⁇ accumulation and infectious virus. It can be explained that it is a process of neuronal function disruption due to the development of pathology such as cell damage caused by T cells that have migrated to the inflammatory site (AD, "HHV-6 encephalitis cascade hypothesis"). And since the hippocampal nerve function has a regenerative ability, it can be expected to compensate for dead cells and repair damaged areas, so early treatment with an antiviral agent having the anti-HHV-6/7 action of the present invention such as foscarnet. If you do, you can expect to be asymptomatic.
  • HHV-6 encephalitis patients (“post-transplant HHV-6 encephalitis patients”) that develop after transplantation of hematopoietic stem cells or organs show neurological symptoms similar to the cognitive impairment observed in patients who developed AD
  • the dosage and administration confirmed to have a significant therapeutic effect on post-transplant HHV-6 encephalitis patients can be referred to.
  • excellent therapeutic results have been reported in patients with HHV-6 encephalitis when given by full-dose (180 mg/kg/day) intravenous infusion for several days.
  • foscarnet has an action of binding to Ca ++, and clinically known side effects such as hypocalcemia.
  • the action of reducing Ca ++ inside and outside the excessive cells and the action of restoring homeostasis (FIG. 5) can be expected.
  • a ⁇ acts as a calcium channel and enhances the influx of excess calcium into cells (A Drews et al., Scientific Reports 6:31910:1-12, 2016)
  • HHV-6 which is the cause of A ⁇ production.
  • Inhibition of /7 reactivation indirectly contributes to the reduction of intracellular Ca ++ .
  • foscarnet In order for foscarnet to exert its antiviral effect on HHV-6/7 reactivated in the brain, it must pass through the BBB and reach the infection site. Foscarnet crosses the BBB and can maintain a concentration that has an effect on HHV-6/7 in cerebrospinal fluid for about 6 hours after administration (Fig. 4), and is therefore suitable for AD treatment.
  • an antiviral agent against HHV-6/7 suppresses at least A ⁇ production, A ⁇ uptake into human nerve cells, oligomer formation, and/or A ⁇ accumulation. Can be treated with antiviral agents effective against HHV-6/7.
  • the main player of HHV-6 encephalitis is HHV-6B, but in the case of AD, it is highly possible that reactivation of HHV-6A and HHV-7 is involved in the spread and exacerbation of the disease, and further HSV- It is known that encephalitis caused by 1 and cytomegalovirus (CMV) infection also exhibits dementia-like symptoms.
  • an antiviral agent aiming at AD treatment has an action not only on HHV-6B but also on HHV-6A and HHV-7, and further has a multi-antiviral action against other herpesviruses such as HSV-1 and CMV. .. Since foscarnet has such multi-antiviral activity, it can be expected to have a therapeutic effect on AD.
  • the present invention relates to a human herpesvirus type 6 (HHV-6A and HHV-6B) and type 7 (HHV-7) and the like, wherein an antiviral agent having an excellent antiviral action is used for treating and preventing AD.
  • Applications include, but are not limited to, the following first through eleventh embodiments.
  • an antiviral agent that can be used for treatment (including prevention) of Alzheimer's disease (AD) is human herpesvirus 6 (HHV-6A and/or HHV-6B) and/or human herpesvirus.
  • Type 7 A drug containing a compound having a strong antiviral action against HHV-7 as an active ingredient, and is preferably effective against herpes simplex virus type 1 (HSV-1), cytomegalovirus (CMV), etc. It is a drug containing a compound having a multi-antiviral action as an active ingredient.
  • the compound having the above-mentioned antiviral activity includes a compound that inhibits the DNA polymerase of herpesvirus, a helicase-primase complex compound essential for the synthesis of viral DAN, a compound that inhibits the packaging of viral DNA into a capsid, and
  • a pyrophosphate analog a compound that directly binds to pyrophosphate of viral DNA polymerase and selectively inhibits DNA synthesis.
  • pyrophosphate analogs do not require activation by phosphatase, are unlikely to develop resistance, and are suitable for AD treatment for long-term administration.
  • Compounds having an antiviral effect other than the pyrophosphate analog can be expected to have an inhibitory effect on virus growth, such as selectively inhibiting viral DNA polymerase.
  • the antiviral effect of the pyrophosphate analog can be complemented.
  • the above-mentioned pyrophosphate analog is phosphonoacetic acid or phosphonoformic acid and/or a derivative thereof, which binds to the pyrophosphate binding site of DNA polymerase and selectively inhibits virus growth, and calcium antagonism.
  • the action and anti-aspartic protease action can be expected.
  • the phosphonoformic acid represented by the following basic skeleton (formula 1) or a salt thereof, or a solvate thereof is referred to as a “phosphonoformic acid derivative”.
  • a fourth form is a phosphonoformic acid derivative, which has a molecular formula of “CNa 3 O 5 P ⁇ 6H 2 O” and is represented by the following (formula 2). Although not limited to the present invention, the compound represented by (Formula 2) is used as the "foscarnet" in this example.
  • the above foscarnet preparation (injection) is sold under the trade name of "Foscavir (registered trademark)" (Japan) or “Foscavir” (US etc.) and is mainly marketed worldwide for indication of cytomegalovirus infection. ing.
  • the fifth mode is a method of using a formulation containing foscarnet as an active ingredient and an oral formulation for the treatment (including prevention) of AD.
  • This use includes treatment of Preclinical AD.
  • Preclinical AD refers to the state from the stage where the accumulation of amyloid ⁇ in the brain was proved to the stage before the initial cognitive impairment (MCI), Stage 1 was the stage where amyloid ⁇ was accumulated in the brain, and Stage 2 was the amyloid.
  • Stage 3 consists of three stages: the accumulation of ⁇ accompanied by degenerative findings in the brain, and the stage where a slight decrease in cognitive function began.
  • Treatment for Preclinical AD preferably begins at Stage 3, more preferably Stage 2.
  • the sixth form is a method in which a foscarnet preparation for treating AD is an injection, and the use thereof is also a preemptive therapy.
  • Preemptive therapy significantly reduces the amount of HHV-6A, HHV-6B and HHV-7 viral DNA in cerebrospinal fluid and/or plasma or blood.
  • the effective dose (60-180 mg/kg/day) of pretreatment with foscarnet is A method of continuous administration by intrathecal administration or intravenous drip infusion for 3 to 10 days, preferably 3 to 5 days, divided into 2 or 3 times a day.
  • the method for inspecting the DNA of each virus may be the method defined in Non-Patent Document 3) or any other method commonly used by medical institutions.
  • the amount of each viral DNA which is a reduction target, can be appropriately modified in consideration of the patient's condition.
  • the duration of preemptive therapy and the dose of foscarnet per day or per day can be adjusted appropriately.
  • the drug can be directly transferred to the infected site, and thus the dose can be further reduced.
  • the seventh form is an injectable foscarnet preparation, which is a maintenance therapy after pre-emptive therapy for the purpose of preventing reactivation of each virus.
  • an effective dose of foscarnet is 60 to 120 mg/ A method in which kg/day is divided into 2 to 3 times a day and administered intravenously, intramuscularly or subcutaneously, preferably intravenous drip for 3 to 6 months.
  • the duration of maintenance therapy and the dose of foscarnet per dose or per day can be appropriately adjusted.
  • the foscarnet preparation is an oral preparation, and with or as an alternative to maintenance therapy by injection, for example, the effective daily dose of foscarnet is 2000 mg to 6000 mg, or 3 to 1 day.
  • a method of maintenance treatment characterized by continuous administration for 3 to 6 months in 5 divided doses. Since foscarnet has mucosal stimulant properties, teprenone, sucralfate, sodium azulensulforate, repamipide, polaprezinc, irsogladine maleate, bexanate hydrochloride, spartalcon, and cetraxate hydrochloride are administered when its oral preparation is administered.
  • a combination with a drug having a gastric mucosa-protecting action such as salt or ecabet sodium hydrate, or a combination thereof is preferable.
  • a drug having a gastric mucosa-protecting action such as salt or ecabet sodium hydrate, or a combination thereof.
  • the duration of maintenance therapy and the dose of foscarnet per dose or per day can be appropriately adjusted.
  • the ninth mode is a combination of a foscarnet preparation and a compound having an antiviral effect having a different chemical or mechanism of action, or a preparation containing the compound, for treating (including preventing) AD. How to use.
  • the antiviral agent used in combination contains nucleoside analogs such as famciclovir (JP 04-275229A), acyclovir (US Pat. No. 4,957,924), valacyclovir (patent 3350055), valganciclovir, penciclovir and brivudine. Formulation, cidofovir (Patent No.
  • a formulation containing a nucleotide analog such as a prodrug or derivative thereof a formulation containing a nucleoside compound, a formulation containing a non-nucleoside DNA polymerase inhibitor compound, helicase/primer Preparation containing ze complex inhibiting compound, compound inhibiting package to viral DNA capsid, anti-CD40 antibody (Japanese Patent Publication No. 2002-543150), PD-1 antibody (Patent No. 5701266), tetrahydro-2H-thiopyran-carboxamide derivative (Patent No. 5011739) and a preparation containing a ghrelin derivative (JP-A-2018-138586), but the preparation is not limited thereto.
  • a nucleotide analog such as a prodrug or derivative thereof
  • a formulation containing a nucleoside compound a formulation containing a non-nucleoside DNA polymerase inhibitor compound
  • a foscarnet preparation is used for treating and preventing AD, and is a drug for treating other AD having a different mechanism of action (AD therapeutic agent), specifically, a factor that worsens AD.
  • AD therapeutic agent a mechanism of action
  • Foscarnet has an action of suppressing excessive A ⁇ production and secretion as an innate immune reaction by suppressing the proliferation of virus causing excessive cell death.
  • foscarnet may exert an anti-aspartic protease action in cells, and it can be expected to suppress the production of A ⁇ .
  • a ⁇ an anti-aspartic protease action
  • Drugs that can be used in combination include drugs that inhibit acetylcholinesterase such as donepezil, drugs that have a nicotinic acid receptor-enhancing effect with acetylcholinesterase inhibition such as galantamine, and both acetylcholinesterase and butyrylcholine esterase such as rivastigmine.
  • drugs that block drugs that block NMD receptors such as memantine.
  • agents that act on secretase that decomposes A ⁇ to inhibit the production of A ⁇ (Tables 2010-5209014 and 2011-518224), agents that inhibit the accumulation of A ⁇ or promote the degradation, or the brain of A ⁇ Agents that promote excretion from the room, agents that inhibit A ⁇ aggregation or A ⁇ protofibril formation (Table 2017-521427), or inhibitors of protein polymerization/activation (Table 2003-528171, Table 2017) -521427), an oxidative stress/endoplasmic reticulum stress-induced apoptosis inhibitor (JP-A 2008-239538), a vaccine (JP-A 2007-522119, JP 2010-522559), and the like can also be used in combination.
  • an excessive calcium influx inhibitor into cells ibuprofen, ginkgo extract, or other anti-inflammatory drug, insulin or other antidiabetic drug, anti-IL-6 antibody, anti-IL-12 antibody, or other inflammatory cytokine It can also be used in combination with a drug that neutralizes chemokines.
  • HHV-6A and HHV-6B of AD patients and HHV-7 virus as a biomarker for confirming the effect of a drug for diagnosing AD and therapeutic agents, viral DNA or antibody or a fragment thereof, saliva, It is a method to detect from blood, plasma, cerebrospinal fluid (CSF), etc.
  • CSF cerebrospinal fluid
  • the most accurate method for confirming viral infection in the brain, especially the hippocampus, is to measure viral DNA in CSF.
  • biomarker for confirming the therapeutic effect of an antiviral agent used for the diagnosis of AD at the risk of suffering from AD or the development of AD in the cerebrospinal fluid or plasma or blood of the subject, Alternatively, viral DNA, protein, glycolipid, or viral composition glycoprotein specific to HHV-6A and HHV-6B and HHV-7 in saliva, preferably in blood or saliva are used. In this case, it can be used in combination with a diagnostic method for AD or a biomarker such as amyloid PET, tau PET, and MRI.
  • Amyloid in CSF, tau protein, amyloid PET test as a biomarker of a method for diagnosing the risk of suffering from AD, diagnosing the progress of AD, and confirming the therapeutic effect of an AD therapeutic drug including an antiviral agent used for AD treatment , Fluoro-D-glucose PET, MRI examination are widely used, and many methods have been developed as biomarkers for confirming the diagnostic and therapeutic effects of AD.
  • a method of using the viral DNAs of HHV-6A, HHV-6B and HHV-7 as a biomarker for AD diagnosis is not known.
  • HBV-6 and HHV-7 are the causative viruses of exanthema subitum, is being prepared by the National Institute of Infectious Diseases (Reference)
  • the real-time PCR method specified in 24 can be used as a standard method.
  • the preparation of foscarnet according to the present invention not only treats AD but also reactivates latently infected herpesviruses, particularly HHV-6 and HHV-7, and/or their proliferation, and accumulation of amyloid ⁇ . , And the antiviral effect of foscarnet can be used as a therapeutic agent for the following diseases caused by viral infection.
  • HHV-6 encephalitis infant
  • idiopathic rash pneumonia, age-related macular degeneration, oral cancer, chronic fatigue syndrome, major depression syndrome, bipolar disorder, heart failure, drug hypersensitivity, idiopathic thrombocytopenic purpura, rosy color Pityriasis, multiple sclerosis, Lewy body degeneration, spinal and cerebellar degeneration, meningitis, Hashimoto's disease, epilepsy, medial temporal lobe sclerosis, frontotemporal lobar degeneration, muscle atrophy lateral sclerosis , Huntington's disease, Rasmussen encephalitis, autoimmune encephalitis, limbic encephalitis, HSV-1 encephalitis, CMV encephalitis, HIV encephalitis, EVB encephalitis, EKV nephropathy, hematopoietic stem cell/bone marrow or hemorrhagic after organ transplantation Use to treat (including preventive) cystitis, interstitial ne
  • the parenteral preparation of foscarnet includes dosage forms for subcutaneous, intramuscular, intrathecal or intravenous administration, infusions, drip packs, nasal administration and the like. Desirable for preemptive therapy is a formulation prepared for intravenous injection capable of high dose administration, and more desirable is a formulation for intravenous drip infusion.
  • a suitable diluent physiological saline, etc.
  • sealed containers such as ampoules, vials, infusion packs, etc. It can be manufactured by filling.
  • the preparation is prepared by a general manufacturing method, excipient, disintegrant, binder, lubricant, suspending agent, tonicity agent, emulsifier, sweetness. It can be produced by mixing foscarnet with an additive such as a coloring agent, a fragrance, or a coloring agent by a conventional method.
  • Foscarnet is a compound with extremely high stability, but it is preferable to use an easily decomposing substance such as starch, lactose, or mannitol as an excipient because it easily chelates metals and has a mucous stimulating property.
  • foscarnet in order to accelerate absorption from the intestinal tract or to gradually release it, foscarnet can be encapsulated in microcapsules by a known method, or tablets having a multi-structure can be formed.
  • a high-dose formulation that exceeds 1 gram as foscarnet conventional tablets and carcasses (including soft capsules) are large in formulation and difficult for the elderly to take. Therefore, granules, fine granules, powders and jelly preparations can also be used.
  • an oral liquid such as a drink
  • distilled water for injection or physiological saline is used as a base
  • foscarnet is dissolved according to a conventional method, and salt or sugar, syrup, etc. are added if necessary, and it is made of glass.
  • foscarnet has mucosal stimulant properties, teprenone, sucralfate, sodium azulensulforate, repamipide, polaprezinc, irsogladine maleate, bexanate hydrochloride, sofarcone, when administering its oral preparation, A combination with a drug having a gastric mucosal protective action such as cetraxate hydrochloride, ecabet sodium hydrate or the like or a combination thereof is desirable.
  • a drug having a gastric mucosal protective action such as cetraxate hydrochloride, ecabet sodium hydrate or the like or a combination thereof is desirable.
  • a mixture of foscarnet and AD agent or gastric mucosa protective agent can be produced in the same manner as foscarnet single agent. Alternatively, it can be produced by mixing microcapsules encapsulating foscarnet with microcapsules (including nanotube capsules) encapsulating AD agent or gastric mucosa protective agent.
  • Diagnosis of AD is divided into three stages, initial (mild), intermediate (medium), and late (advanced) according to the symptoms, and existing AD treatments have also acquired indications according to this stage.
  • Foscarnet can be used at any stage of AD in terms of mechanism of action. From the recommended dosage of drip infusion, it is a drug suitable for treatment of severe AD, but from the experience of treatment for HHV-6 encephalitis, administration at an early or mild stage stops AD progression and recovery. It is desirable because it can be expected. This allows the use of foscarnet for Preclinical AD.
  • Preclinical AD refers to the state from the stage where A ⁇ accumulation is proved in the brain to the stage before early cognitive impairment (MCI).
  • Stage 1 is the stage where A ⁇ accumulation is observed in the brain, and stage 2 is A ⁇ accumulation In addition to stage 2, stage 3 is accompanied by degenerative findings in the brain, and in addition to stage 2, a slight decline in cognitive function begins.
  • treatment for Preclinical AD is preferably initiated at stage 3, more preferably stage 2, foscarnet is also suitable for treatment at this time.
  • biomarkers for AD the ratios of A ⁇ and A ⁇ 42 to other A ⁇ in cerebrospinal fluid (CSF), tau protein, amyloid PET test, fluoro-D-glucose PET, MRI test, etc. are widely used.
  • CSF cerebrospinal fluid
  • tau protein tau protein
  • amyloid PET test fluoro-D-glucose PET
  • MRI test etc.
  • these methods cannot identify the presence or type of infectious virus that is the main cause of AD.
  • the real-time PCR method defined in "Pathogen Inspection Manual" (reference document 24) can be used as a standard method.
  • the present invention is intended to treat AD by suppressing the reactivation of latent HHV-6/7, and the cerebrospinal fluid capable of measuring the DNA level of HHV-6/7 in the brain in a relatively minimally invasive manner.
  • the DNA level of HHV-6/7 in CSF is treated as an index of preventive and therapeutic effects.
  • the HHV-6A and HHV-6B and HHV-7 viral DNA in CSF of AD patients are below the detection limit.
  • AD although it is certainly common with HHV-6 encephalitis as a cognitive mechanism lowering symptom, unlike HHV-6/7 high-concentration infection/highly active HHV-6 encephalitis, it is considered to progress mildly. Has been. Therefore, the dosage regimen of foscarnet in treating AD is likely to be fairly modest. The latent form of HHV-6 cannot completely suppress its reactivation. It is also possible to select a dosage and a method for preservative treatment, in which the amount of viral DNA in the healthy person or in the cerebrospinal fluid is below the detection limit, and then the patient is switched to maintenance therapy to improve patient compliance.
  • the "effective amount” is a dose required to at least not increase the infection level of HHV-6/7 in cells of the cranial nervous system, and the infection level is HHV-6/7-derived DNA in CSF. It can be evaluated as the accumulation level of.
  • the specific dose varies depending on the condition and symptoms of viral infection in the patient, physical condition, administration route, efficacy of antiviral agent against each virus, and the like.
  • the post-onset full dose (180 mg/kg/day) intravenous infusion shows an excellent effect, for example, for AD patients
  • a 250 mL vial (24 mg/mL) injection (“Foscavir (registered trademark) injection” formulation) containing 6 g of foscarnet sodium hydrate as an active ingredient is used as a preemptive agent.
  • the maximum effective dose for the first dose of preemptive therapy is 180 mg/kg/day as a standard, but the effective dose may be adjusted appropriately if it is administered intrathecally in patients with renal impairment or to ensure compliance.
  • an oral preparation containing 500 mg to 1500 mg of foscarnet and use it for maintenance therapy after discharge.
  • 2000 to 6000 mg of foscarnet is administered in 3 to 4 divided doses daily. The period is until the symptom of AD (especially cognitive impairment) disappears, but it is expected to be 3 to 6 months.
  • AD especially cognitive impairment
  • foscarnet has mucosal stimulant properties, teprenone, sucralfate, sodium azulensulforate, repamipide, polaprezinc, irsogladine maleate, bexanate hydrochloride, sofarcone, when administering its oral preparation, It is desirable to use in combination with a drug having a gastric mucosa-protecting action such as cetraxate hydrochloride and ecabet sodium hydrate, or a combination thereof.
  • a drug having a gastric mucosa-protecting action such as cetraxate hydrochloride and ecabet sodium hydrate, or a combination thereof.
  • the DNA amount of HHV-6A, HHV-6B and HHV-7 in cerebrospinal fluid, plasma or saliva should be monitored regularly (once every 1 to 3 months), and if it exceeds the standard, it should be checked again. After being hospitalized, administer foscarnet according to pre-emptive therapy. The dose should be adjusted appropriately according to the condition of the patient and the amount of virus detected.
  • AD occurs in the elderly, and renal function is often reduced in the elderly. For high doses, it is desirable to reduce the risk of renal damage by diuretics and adequate water supplementation after administration. Further, when renal damage is a concern, it is desirable to change the dose of foscarnet according to the value of urinary creatine, in addition to the method of reducing the dose by intrathecal administration.
  • the method of changing the dose is routine and is described in detail in the treatment guideline for HHV-6 encephalitis (reference document 32). This guideline can also be applied to foscarnet therapy for AD.
  • Foscarnet has extremely high anti-HHV-6 activity as compared with other antiviral agents, and has the greatest difference between the effective dose and the toxic dose, and is excellent in safety.
  • the antiviral assay was carried out by infecting each cell with 100 CCID 50 of HHV-6 per 10 6 cells, culturing the cells in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine, and 0.1% sodium bicarbonate at 37°C. Absorbed for 90 minutes. After washing and cell number adjustment, the adjusted antiviral agents were serially diluted and added. The medium was replaced with an antiviral agent-added medium every 3 to 4 days, and the concentration of the antiviral agent in which the viral growth was suppressed to 50% was calculated on the 10th to 12th day and defined as CC 50 . (Non-patent document 16)
  • foscarnet has an antiviral effect against HHV-6B in an effective dose about twice that of HHV-6A and HHV-7, and it is not necessary to significantly change the dose for each target virus, and to maintain the balance. It is a taken antiviral agent (Fig. 3).
  • Foscarnet has an antiviral effect against the following various viruses (Non-patent document 17).
  • CMV, HSV-1, EBV, HIV-1 and the like are known to have latent infection in the brain, and when activated, they are known to cause encephalitis. Therefore, it is desirable that an antiviral agent as a therapeutic agent for AD has an effect against such a virus (Clinigen Co., Ltd., “Foscavir (registered trademark) Interview Form March 2019 edition”, 2019).
  • Example 1 Inhibitory effect of foscarnet on intracellular uptake of amyloid ⁇ (including oligomer) in virus-(HHV-6, HHV-7)-infected human-derived neurons
  • amyloid ⁇ A ⁇
  • the procedure of this test was as follows. (1-1) Preparation of HHV-6A, HHV-6B and HHV-7 Virus Solutions Each virus solution used in the test was prepared according to the following test materials and procedures (a) Test material 1) Used for the test The following cell lines were provided by the HHV-6 Foundation (Santa Barbara, California, USA).
  • ⁇ HSB-2 strain HSB-2 Uninfected Cell, Master Seed, 1.0 mL, @1 ⁇ 10 7 cells, 12/17/2018, (BIOCELL Diagnostics H10-865); 10% FBS, 1 ⁇ ATB added IMDM medium (Reference: CCRF-HSB-2(ATCC CCL-120.1) Product Sheet)
  • ⁇ Sup-T1 strain Sup-T1 Uninfected Cell, Master Seed, 1.0 mL, @1 ⁇ 10 7 cells, 12/17/2018, (BIOCELL Diagnostics H10-864); RPMI- supplemented with 10% FBS, 1 ⁇ ATB Culture using 1640 medium (reference: Sup-T1(ATCC CRL-1942) Product Sheet)
  • ⁇ HHV-6A/HSB-2 strain Master Seed, HHV-6A, GS strain, infected HSB-2 cells, 1.0 mL, @5 ⁇ 10 6 cells/mL, 8/13/2018, (BIOCELL Diagnostics H10-849 ); Culture in the same medium as HSB
  • 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)
  • non-infected HSB-2 strain for HHV-A and Sup-T1 strain for HHV-6B and HHV-7 were cultured by the following procedure. 1) A tube of cryopreserved cells was rapidly thawed in a warm bath at 37°C, 10% FBS for HSB-2 cells and IMDM medium supplemented with 1xATB, and 10% FBS, @1x for Sup-T1 cells. Mix with RPMI-1640 medium (9 mL each) supplemented with ATB.
  • HHV-6A/HSB-2 HBV-2 strain infected with HHV-6A
  • HHV-6B/Sup-T1 Sup-T1 strain infected with HHV-6B
  • HHV-7/Sup-T1 Cryopreserved cells (5 ⁇ 10 6 cells each) of Sup-T1 strain infected with HHV-7 were rapidly thawed in a warm bath at 37°C.
  • Infected live cell count Infected cell viable cell count x (ratio of infected cell >12 ⁇ m viable cell-non-infected cell >12 ⁇ m viable cell)
  • Number of dead cells specific to infected cells total number of infected cells x (survival rate of uninfected cells-survival rate of infected cells)
  • Infection rate (%) (number of infected cells + number of dead cells specific to infected cells) / total number of infected cells x 100
  • (E) Preparation of virus solution (e-1) Procedure HSB-2 or Sup-T1 (equivalent to about 5 times) is added together with an appropriate amount of fresh medium and subcultured to increase the infection rate to about 60 to 80%. Continue culturing until. Then, collect the cells together with the medium in a 50 mL centrifuge tube and store them frozen (-80°C). Then, a virus solution is prepared by the following procedure. 1) Heat thaw at 37°C for rapid thawing and mix for 30 seconds with a vortex mixer. After cryopreservation, quickly thaw and mix once more with a vortex mixer.
  • SN2 and SN3 are mixed and centrifuged (4,500 rpm, 20 min, 4°C), and the supernatant is collected (this is designated as SN4). 8) After filtering 0.45 ⁇ m of SN4 through a filter, concentrate it with a centrifugal ultrafiltration filter. Aliquot 200 to 300 ⁇ L and store frozen (-80°C: virus solution)
  • HHV-6 PCR product with known concentration (HHV-6B as a template, PCR purified with HHV6-orf67-F1 primer and HHV6-orf67-R1 primer by column purification) or HHV-7 PCR product (HHV- 7 as a template, Hs-ACTB-GF1 primer and Hs-ACTB-gR1 that the product was PCR amplified with primers were column purified)
  • dilution series is also in parallel to perform the Real -time PCR analysis, calibration curve (C 1 Create the relationship between the value and the number of template DNA (virus genome copy number: GC). Calculate the GC in each virus solution based on the calibration curve. The results are shown in (Table 11) below.
  • E-3) Measurement of titer The titer of virus in infected cells was measured as follows. 1) Prepare uninfected HSB-2 (for HHV-6A) or Sup-T1 (for HHV-6B and HHV-7) at 3.3 ⁇ 10 5 cells/mL and seed 0.45 mL/well on a 24-well plate. .. Overnight culture. 2) preparing a diluted solution with 2 0-2 -5 was diluted with medium each virus solution 50 [mu] L. 3) Add 50 ⁇ L of serially 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) The virus titer (IFU/mL) was determined by calculating the infection rate calculated by the procedure specified in the above “Addition of A ⁇ 42 and virus infection”.
  • GC virus genome copy number
  • 1) Prepare genomic DNA from 50 ⁇ L of virus solution.
  • DNeasy Blood & Tissue Kit Purification of Total DNA from Animal Blood or Cells (Spin-Column Protocol)]
  • prepare the sample including RNase A treatment until just before column addition prepare the sample including RNase A treatment until just before column addition.
  • a ⁇ 42 for the test was prepared by the procedure described in 1.
  • a ⁇ 42 powder (Amyloid ⁇ -Protein (Human, 1-42), 0.59 mg; MW 4514.0; lot 680405, Peptide Institute, 4349-v) was allowed to stand at room temperature for 30 minutes.
  • HFIP (1,1,1,3,3,3 Hexafluoro) with a syringe connected to an injection needle in the draft chamber.
  • DMSO Dimethyl sulfoxide: lot LKF233. Fujifilm Wako 037-24053
  • a long-wave sonication device Bioruptor UCD-250, S/N 250581, Sonic Bio Co., Ltd. disrupts and lyses cells by sonication at 160 W for 10 minutes, 10 sec On/10 sec Off/30 cycles.
  • test sample The following was set as a sample used for the test.
  • a ⁇ 42 is 0.1 to 1,000 nM (two-step dilution with a common ratio of 10) and 6 conditions without addition, foscavir (registered trademark) is 800 ⁇ M final concentration and 2 conditions without addition, virus solution is HHV-7 only, HHV-7 And HHV-6A were mixed, HHV-7 and HHV-6B were mixed, and no addition was carried out under MOI 0.1 and 1.0 conditions, respectively, and a sample treatment group was set as shown in (Table 12) below.
  • a ⁇ 42 was added to the medium of the test cells and the virus was infected to the test cells.
  • FCN foscavir (registered trademark) injection 24 mg/mL for intravenous infusion, serial number XX26, containing 6 g of foscarnet as an active ingredient.
  • FCN foscavir (registered trademark) injection 24 mg/mL for intravenous infusion, serial number XX26, containing 6 g of foscarnet as an active ingredient.
  • HHV-6A, HHV-6B and HHV-7 can infect test cells by genetic test alone, but also HHV-6A and HHV-7 or combined infection of HHV-6B and HHV-7. Was observed.
  • HHV-6A and HHV-7 have high concentrations of GC in both and are considered to be compatible with each other.
  • the GC of HHV-6B was remarkably low. Against this background, it was suggested that HHV-6B had low infectivity to test cells.
  • Example 3 In the presence of amyloid ⁇ (including oligomer), foscarnet inhibits calcium uptake into nerve cells After culturing SH-SY5Y cells on a 96-well microplate, only A ⁇ was added to the culture solution. , And A ⁇ and foscarnet are added, and the cells are further cultured for 1 to 3 days. After removing the culture solution so as not to damage the cells and washing, a fluorescent substrate solution is added and the intracellular calcium amount is measured by fluorescence intensity measurement.
  • amyloid ⁇ including oligomer
  • Example 4 Anti- ⁇ secretase action of foscarnet Since foscarnet has antimetal (zinc) enzyme inhibitory activity, it inhibits the activity of ⁇ -secretase, which is an aspartic protease (having zinc at the active site). Whether or not it has an effect was verified using the following test.
  • foscavir registered trademark
  • foscavir registered trademark
  • serial number XX26 containing 6 g of foscarnet as the main component
  • Fluorimetric TM model number AS-71144
  • the positive control inhibitor LY2886721 attached to the kit and the commercially available CEM inhibitor ADZ3839 (free base) (CS-5933) were used.
  • Example 5 Killing/effect of HHV-6/7 on infected cells Obtained by the above procedure on non-infected cells (HSB-2 for HHV-6A, Sup-T1 for HHV-6B and HHV-7)
  • the virus solution was added, and the presence or absence of virus infection and changes in the morphology of infected cells were observed as follows.
  • (5-1) Regarding HHV-6A a medium for culturing only HSB-2 (uninfected strain) and a medium for culturing uninfected strain are added with the HHV-6A virus solution obtained by the above procedure, Sampling was performed and the morphology of cells in each medium was observed with an optical microscope (Olympus CKX 41).
  • FIG. 10-1 uninfected cells
  • the shape and size of each cell were uniform, and almost no dead cells were observed.
  • FIG. 10-2 HHV-6A added
  • enlarged cells and dead cells are scattered.
  • HHV-6B and HHV-7 a medium for culturing only SupT-1 (uninfected strain) and a medium for culturing an uninfected strain are used in the HHV-6B virus solution or HHV- 7
  • the virus solution was added, and the sampled medium was observed with an optical microscope (Olympus CKX 41) 20 times to observe the morphology of the cells in each medium, and the Leica MC 120 HD was used for automatic exposure mode and taken at a resolution of HD1080-50. did.
  • FIG. 11-1 cells not infected with virus
  • dead cells were not seen, and the cell shape and size were uniform.
  • FIG. 11-2 HHV-B added
  • FIG. 12 HHV-7 added
  • HHV-6A and HHV-6B which are latently infected with nerve cells and glial cells in the hippocampus in which the neuron is newly formed in AD, undergo cell death including apoptosis of host cells.
  • T cells that have migrated to the site of inflammation also induce necrosis, and various inflammatory substances (eg, HMGB-1, TDP-43 as DAMPs (Damage/ Danger Associated Molecular Patterns)) are generated from the cells.
  • various inflammatory substances eg, HMGB-1, TDP-43 as DAMPs (Damage/ Danger Associated Molecular Patterns)
  • GSK-3 ⁇ , glutamic acid, Ca ++, etc. are released to enhance inflammation, which leads to various pathologies associated therewith, especially excessive death of nerve cells (Non-Patent Document 18 and Q Chen et. al., Signal Transduction and Targeted Therapy, 3(18): 1-11, 2018).

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Abstract

The present invention provides a novel drug useful for fundamental therapy and prevention of Alzheimer's disease (AD). As a pharmaceutical composition for treating or preventing AD, a drug containing, as an active ingredient, a compound having anti-viral activity against HHV-6A, HHV-6B, and/or HHV-7, particularly foscarnet, is used. The drug does not only stop the progression of AD, but also is likely to serve as a fundamental therapeutic means. In addition, by using, as an AD biomarker, DNA or proteins of HHV-6A, HHV-6B, and/or HHV-7 derived from a subject's cerebrospinal fluid (CSF), blood plasma, blood, or saliva, or antibodies specific to HHV-6A, HHV-6B, and/or HHV-7 or fragments of the antibodies, the risk of developing AD or the state of progression of AD can be diagnosed, or the therapeutic effect of an antiviral agent used in the treatment of AD can be confirmed.

Description

アルツハイマー病を治療又は予防する為の抗ウイルス剤及びその使用Antiviral agent for treating or preventing Alzheimer's disease and use thereof
　本発明は、脳神経疾患、特にアルツハイマー病に対する治療薬及びその使用に関する。 The present invention relates to a therapeutic agent for cranial nerve diseases, particularly Alzheimer's disease and its use.
（アルツハイマー病の現状と病態）
　アルツハイマー病（「AD」）は認知症の大半を占める疾患で、2015年の全世界の患者数は約3000万人、これが2050年には1億人を超えるとの予想もある。
　ADは段階的に症状が進行し、記憶障害の進行に加えて手段的日常生活動作（「ADL」）や基本的ADLに障害が出ると中期又は中等度を診断される。また、AD患者では早期に嗅覚障害が生じている。後期になると重度のADLの低下に伴い接触・嚥下障害や肺炎、転倒・骨折などを併発し、終末期を迎える。尚、中期と後期の間で様々な周辺状況が出現することがあり、不安、抑うつ、不眠、興奮、易怒性、徘徊、幻覚、妄想などが指摘されている。なお、AD患者の脳でのAβの蓄積（老人斑の出現）は、発症の１５年以上も前から認められる現象であるが（参考文献１）、その成因は明らかになっていない。
　参考文献１：RJ Bateman et al., N. Engl. J. Med. 367 (9): 795-804, 2012 (Current status and pathology of Alzheimer's disease)
Alzheimer's disease (“AD”) is a disease that accounts for the majority of dementia, and the number of patients worldwide in 2015 was about 30 million, and it is predicted that by 2050, it will exceed 100 million.
AD develops a gradual symptom and is diagnosed as having a middle stage or moderate degree when a disorder of the instrumental activities of daily living (“ADL”) or basic ADL occurs in addition to the progress of memory disorder. In addition, AD patients have an early olfactory disorder. In the second half, severe ADL decline causes contact and dysphagia, pneumonia, falls and bone fractures, and the end of life is reached. In addition, various peripheral situations may appear between the middle stage and the latter stage, and anxiety, depression, insomnia, excitement, irritability, wandering, hallucinations, delusions, etc. have been pointed out. The accumulation of Aβ (the appearance of senile plaques) in the brains of AD patients is a phenomenon that has been observed for more than 15 years before the onset (Reference Document 1), but the cause has not been clarified.
Reference 1: RJ Bateman et al., N. Engl. J. Med. 367 (9): 795-804, 2012.
（アルツハイマー病の原因）
　AD患者の脳内では、アミロイドβタンパク質（「Ａβ」）の増加とその集合体（「Aβオリゴマー」）の蓄積が顕著で、Aβ（特に４２個のアミノ酸からなる可溶性の高いAβ42）を主成分とする老人斑の増加、脳アミロイド血管症などがみられる。このAβ・Aβオリゴマーの増加と蓄積によりシナプスの脱落、ミクログリアの活性化、IL-6やIL-12、TNF-αなどの炎症性サイトカインやCCL2などのケモカインの高発現、脳実質のカルシウム濃度の上昇、グルコース代謝の異常などが起こり、神経細胞壊死を誘発するなど、AβはAD発症のキーファクターと考えられている（非特許文献１、参考文献２，３）。　
　AD患者では脳血管にAβが付着して炎症を生起し、アミロイド血管症を惹起していることが多い（参考文献３）。脳でのアミロイド血管症はBBBの脆弱化や破綻を招き（参考文献４）、末梢血中のT細胞やNK細胞の炎症部位への遊走を促進する。
　また、ADでは微小管を組成する主成分であるタウタンパク質の過剰リン酸化と凝集、それに伴う神経原線維変化、神経細胞死が進み、症状が進行した患者では脳（特に海馬）の萎縮や大量の神経細胞死などが認められる（参考文献５）。
　なお、ADにおける過剰ともいえる細胞死の原因は、細胞内に蓄積したAβ（特にAβ42を主な組成とするオリゴマー）の病理としての「アポトーシス」と考えられている（参考文献１，５）。この細胞死を示す証左としてAD患者の脳内で大量のDNA断片が見られ（参考文献５）、また、海馬での神経細胞の新生が劇的に低下していることが報告されている（非特許文献２）。
　この神経細胞の大量死と新生不全がADにおける病理の根本的な原因といえるが、神経細胞の大量細胞死と新生不全との関係は十分に解明されていない。
　参考文献２：S. West et al., Br. J. Clin. Pharmacol. 80 (2) 221-234, 2015
　参考文献３：B Mroczko et al., International Journal of Molecular Sciences (19): 1-29, 2018
　参考文献４：KA Jellinger et al., Journal of Alzheimer’s Disease 3 (2001):31-40,2001
　参考文献５：上野正樹、「臨床神経学」57巻3号、95－109、2017 (Causes of Alzheimer's disease)
In the brains of AD patients, there is a marked increase in amyloid β protein (“Aβ”) and accumulation of its aggregates (“Aβ oligomers”), and mainly Aβ (particularly highly soluble Aβ 42 consisting of 42 amino acids) Increasing senile plaques as a component and cerebral amyloid angiopathy are seen. This increase and accumulation of Aβ and Aβ oligomers results in synaptic loss, microglial activation, high expression of inflammatory cytokines such as IL-6, IL-12, TNF-α, and chemokines such as CCL2, and brain parenchymal calcium concentration. Aβ is considered to be a key factor for the onset of AD, such as elevation and an abnormality in glucose metabolism, which induces neuronal necrosis (Non-patent Document 1, References 2 and 3).
In AD patients, Aβ adheres to cerebral blood vessels to cause inflammation and often cause amyloid angiopathy (reference document 3). Amyloid angiopathy in the brain leads to weakening and disruption of BBB (reference document 4) and promotes migration of T cells and NK cells in peripheral blood to inflammatory sites.
In AD, hyperphosphorylation and aggregation of tau protein, which is the main component of microtubules, accompanying neurofibrillary tangles, and neuronal cell death, and in patients with advanced symptoms, brain (especially hippocampus) atrophy and large amounts. Neuronal cell death is observed (Reference 5).
The cause of cell death, which can be said to be excessive in AD, is considered to be "apoptosis" as a pathology of Aβ (in particular, an oligomer mainly composed of Aβ42) accumulated in cells (References 1 and 5). As a proof of this cell death, a large amount of DNA fragments were found in the brain of AD patients (reference document 5), and it was reported that neuronal neogenesis in the hippocampus was dramatically reduced ( Non-Patent Document 2).
It can be said that the mass death and neoplasia of nerve cells are the fundamental cause of the pathology in AD, but the relationship between mass death of nerve cells and neoplasia is not 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" Vol. 57, No. 3, 95-109, 2017
（AD薬の開発標的）
　AD発症に脳室内でのAβ（そのオリゴマーを含む）の増加や蓄積あるいはタウタンパク質の変性が関与しているとの観点からAβあるいはタウタンパク質を標的としたAD治療薬の研究・開発が世界的な規模で行われてきた（非特許文献３、参考文献６）。具体的には、Ａβの形成・沈着、Aβ産生酵素であるβセクレターゼ（BASE-1）及びγセクレターゼ、Aβオリゴマー形成、タウタンパク質のリン酸化などを標的とし、その一部はAD疾患モデル動物を用いた試験で効果が示されている。
　ただAβを標的としたAD新薬の多くは有効性や安全性の問題などで開発に成功しておらず、新たな視点、例えば疾患修飾療法療薬（DMTs、Disease-Modifying Therapies ）あるいは最新の知見（iPS細胞など）に基づくAD薬の開発が成されている（非特許文献３）。その中にDMTsの範疇に入るが、ADを慢性炎症性の疾患と捉え、神経組織において炎症をもたらす因子を標的とする創薬（参考文献６）もある。しかし、ADに対するDMTs薬として製品化に至ったものはない。
　参考文献６：AA Fabregat et al., CNS Drugs (2017) 31: 1057-1082, 2017 (Target for AD drug development)
From the perspective that the onset and accumulation of Aβ (including its oligomers) in the ventricles or the denaturation of tau protein are involved in the onset of AD, the research and development of AD therapeutic drugs targeting Aβ or tau protein is worldwide Has been performed on a large scale (Non-patent document 3, Reference document 6). Specifically, it targets Aβ formation/deposition, Aβ-producing enzymes β-secretase (BASE-1) and γ-secretase, Aβ oligomer formation, and tau protein phosphorylation, and some of them target AD disease model animals. The tests used show an effect.
However, many new AD drugs targeting Aβ have not been successfully developed due to issues such as efficacy and safety, and new perspectives such as disease-modifying therapies (DMTs, Disease-Modifying Therapies) or the latest findings ( Development of AD drugs based on iPS cells and the like) has been made (Non-Patent Document 3). Among them, there is a drug discovery (Reference 6), which belongs to the category of DMTs, but considers AD as a chronic inflammatory disease and targets a factor that causes inflammation in nerve tissue. However, no DMTs drug for AD has been commercialized.
Reference 6: AA Fabregat et al., CNS Drugs (2017) 31: 1057-1082, 2017
　ADの発症やその悪化に脳内で起きている慢性炎症が関与していることは定説となりつつあるが、その原因となる因子としてDAMPs（Damage or Danger-Associated Molecular Patterns）が指摘されている（非特許文献４、５）。
　DAMPsは何らかの理由で傷害を受け死亡した細胞から放出される物質群で、その出所として核、細胞質、ミトコンドリア、小胞体などがあり、放出される物質も多種多様である（非特許文献５，参考文献７）。その中でADに関係があるものとして、Aβ、カルシウム（参考文献８）、HMGB-1（参考文献９） 、TDP-43（参考文献１０）、GSK-3β（参考文献１１）、グルタミン酸／グルタミン（参考文献１２） 、ATP（参考文献１３） 及び細菌・ウイルス（参考文献１４，１５）などが俎上に挙がっている。尚、ウイルス等の病原菌の感染に起因するものはPAMPsとも呼ばれている（参考文献７）。
　DAMPsの多くは、細胞死がプログラムされた仕組みで生じる「アポトーシス」ではなく、何らかの病的な要因で生じる「ネクローシス」の際に細胞外に放出される。また、ウイルスに感染した細胞が発現するケモカイン（CCL2/MCP-1など）やサイトカイン（IFN-γ、IL-６、TNFα等）もDAMPs（例えばHMGB-1など）が引き金となることが知られている（非特許文献６）。
　参考文献７：吉田恵次、「外科と代謝・栄養」48巻6号：247‐249、2014
　参考文献８：Y Wang et al., J. of Alzheimer’s Disease & Parkinsonism,7 (5): 1-15, 2017　
　参考文献９：K Fujita et al., Scientific Reports 6 (31895): 1-15, 2016　　
　参考文献１０：XL Chang et al., Molecular Neurobiology 53 (5): 3349-3359, 2016　
　参考文献１１：ML Martin et al., frontiers in Molecular Neuroscience 7 (46): 1-11, 2014　　
　参考文献１２：C Madeira et al., frontiers in Psychiatry 9 (561): 1-9, 2018
　参考文献１３：斎藤秀俊他、「日薬理誌」136：93-97、2010
　参考文献１４：M Sochocka et al., Current Neuropharmacology, 15 (7): 996-1009, 2017
　参考文献１５：射場敏明、「外科と代謝・栄養」51 (1)：1-7、2017 It is becoming established that chronic inflammation occurring in the brain is involved in the onset and exacerbation of AD, but DAMPs (Damage or Danger-Associated Molecular Patterns) have been pointed out as the causative factor ( Non-Patent Documents 4 and 5).
DAMPs are a group of substances released from cells that have been injured and died for some reason, and have sources such as the nucleus, cytoplasm, mitochondria, and endoplasmic reticulum, and the substances released are also diverse (Non-Patent Document 5, Reference Reference 7). Among them, those related to AD include Aβ, calcium (reference document 8), HMGB-1 (reference document 9), TDP-43 (reference document 10), GSK-3β (reference document 11), glutamic acid/glutamine. (Reference 12), ATP (Reference 13), bacteria and viruses (References 14 and 15), etc. are listed. Those caused by infection with pathogenic bacteria such as viruses are also called PAMPs (reference document 7).
Many DAMPs are released extracellularly during "necrosis", which is caused by some pathological factor, rather than "apoptosis," which is a mechanism in which cell death is programmed. It is also known that chemokines (such as CCL2/MCP-1) and cytokines (such as IFN-γ, IL-6 and TNFα) expressed by virus-infected cells are triggered by DAMPs (such as HMGB-1). (Non-patent document 6).
Reference 7: Eiji Yoshida, "Surgery and Metabolism/Nutrition" Vol. 48, No. 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: Hidetoshi Saito et al., "Nippon Pharmacology" 136:93-97, 2010.
Reference 14: M Sochocka et al., Current Neuropharmacology, 15 (7): 996-1009, 2017
Reference 15: Toshiaki Range, "Surgery and Metabolism/Nutrition" 51 (1): 1-7, 2017
（ADと神経細胞死）
　以前は、成人すると神経細胞は新生しないとされていた。しかし、現在では、海馬歯状回や脳後室下部に神経幹細胞が存在し、生涯にわたり神経細胞が新生することが明らかになっている（非特許文献７）。このことは、ADの治療において、成熟を終え、やがてアポトーシスする神経細胞の延命を図るより、神経幹細胞や分化途中の細胞の死を抑制することの重要さを示している。
　尚、脳後室下部に存在する神経幹細胞は神経細胞に分化後、嗅球に移行することは知られているが、脳の他の組織、例えば大脳新皮質の神経細胞となるか否かは不明である。 (AD and nerve cell death)
In the past, it was said that nerve cells do not form in adults. However, it is now clear that neural stem cells are present in the dentate gyrus of the hippocampus and in the lower part of the posterior ventricle of the brain, and neural cells are born throughout life (Non-Patent Document 7). This indicates the importance of suppressing the death of neural stem cells or cells in the process of differentiation rather than prolonging the life of nerve cells that have completed maturation and then apoptotic in the treatment of AD.
Although it is known that neural stem cells existing in the lower part of the posterior part of the brain are differentiated into neurons and then migrate to the olfactory bulb, but it is unknown whether they become neurons in other tissues of the brain, for example, neocortex of the cerebrum Is.
（認知症の原因としてウイルス感染）
　認知症の発症やその悪化にウイルスや真菌などの関与を指摘する報告があり（参考文献１３）、特に単純ヘルペスウイルス１型（HSV-1）が脳内で再活性化したときに惹起する重篤なヘルペスウイルス脳炎（HSVE）の臨床像がADに近いなどの理由で、HSV-1は認知症、特にADの原因の有力な候補とされ（非特許文献８，９、参考文献１７）、また、HSV-1感染時に抗ウイルス剤で治療した患者は、しなかった患者に比べ認知症発症が少なかったとされる研究（参考文献１８）もある。
　しかし、HSV-1がAD発症のリスクファクターであり、HSV-1感染がAβの蓄積やタウ蛋白の過剰なリン酸化を招くことなどは10数年以上前から指摘されているが（非特許文献８）、未だにHSV-1がAD発症の主因との証明は得られていない。
　参考文献１７：SA Harris et al., Frontiers in Aging Neuroscience, 10 (48): 1-24, 2018
　参考文献１８：Tzeng NS. et al., Neurotherapeutics, 15 (2): 417-429, 2018 (Viral infection as a cause of dementia)
There is a report that points out the involvement of viruses and fungi in the development of dementia and its exacerbation (Reference 13), and in particular, the herpes simplex virus type 1 (HSV-1) causes a significant increase in reactivation in the brain. HSV-1 is considered to be a strong candidate for the cause of dementia, particularly AD, because the clinical picture of severe herpesvirus encephalitis (HSVE) is close to AD (Non-patent Documents 8, 9 and Reference 17), In addition, there is also a study (reference document 18) that patients treated with antiviral agents at the time of HSV-1 infection had less onset of dementia than patients who did not.
However, HSV-1 is a risk factor for AD development, and it has been pointed out that HSV-1 infection leads to Aβ accumulation and excessive phosphorylation of tau protein for more than 10 years (non-patent document 8) The proof that HSV-1 is the main cause of AD has not been obtained yet.
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
　HSVE以外にADに似た臨床像を示す潜伏ウイルスの再活性化を原因とする疾患に「HHV-6脳炎」があり、その主たる原因ウイルスはヒトヘルペスウイルス6型B（HHV-6B）である（参考文献１９）。
　HHV-6脳炎は、乳幼児期の初感染時あるいは造血幹細胞や骨髄、臓器の移植時のような免疫力が低下した状況下において、脳内で潜伏感染しているHHV-6（特にHHV-6B）の再活性化が亢進（ウイルスの大量増殖）した場合に発症する（参考文献２０～２２）。しかし、健常人での該脳炎の発症事例は殆ど知られていない。このことは、健常人の脳組織ではHHV-6（A及びB）が再活性化してもウイルスの増殖は限定され、検知でき且つ健康状態に疑念を抱かせる臨床症状を生起しないことを示唆している。
　尚、骨髄や臓器移植後に発症した脳炎がHHV-6（A及びB）に起因するか否かの検査は、他の原因ウイルス等の存在が否定できたとき、脳脊髄液中におけるHHV-6（A及びB）に特異的なDNAの有無及びその数をPCR法で検査することで判定できる（参考文献２４）。
　参考文献１９：河村吉紀他、「日本臨床」69巻3号、423-427、2011
　参考文献２０：A Ansari et al., Emerging Infection Disease 10 (8), 2004
　参考文献２１：緒方正男、「臨床血液」57巻３号:298-306、2016
　参考文献２２：LD Bolle et al., Clinical Microbiology Reviews, Jan: 217-245, 2005
　参考文献２３：J Ongradi et al., J. Neurovirol. 23: 1-19, 2016
　参考文献２４：多屋響子他、国立感染症研究所　病原体検査（検出）マニュアル「突発性発疹」、http://www.niid.go.jp/niid/ja/labo-manual.html In addition to HSVE, "HHV-6 encephalitis" is a disease caused by reactivation of latent virus showing a clinical picture similar to AD, and the main causative virus is human herpesvirus 6B (HHV-6B). (Reference 19).
HHV-6 encephalitis is HHV-6 (especially HHV-6B) that is latently infected in the brain under the condition of weakened immunity such as initial infection in infancy or transplantation of hematopoietic stem cells, bone marrow, and organs. It develops when the reactivation of erythrocytes is accelerated (mass multiplication of virus) (references 20 to 22). However, few cases of the onset of the encephalitis in healthy people are known. This suggests that the reactivation of HHV-6 (A and B) in normal human brain tissue limits the growth of the virus and does not result in any detectable and suspicious clinical signs of health. ing.
It should be noted that the examination of whether or not encephalitis developed after bone marrow or organ transplantation is caused by HHV-6 (A and B) was carried out when the presence of other causative viruses could be ruled out. The presence or absence of DNA specific to (A and B) and the number thereof can be determined by testing by PCR (Reference 24).
Reference 19: Yoshinori Kawamura et al., "Japanese Clinical Practice," Vol. 69, No. 3, 423-427, 2011.
Reference 20: A Ansari et al., Emerging Infection Disease 10 (8), 2004
Reference 21: Masao Ogata, "Clinical Blood," Vol. 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: Kyoko Taya et al., National Institute of Infectious Diseases, Pathogen Detection (Detection) Manual, "Examination of Rash", http://www.niid.go.jp/niid/ja/labo-manual.html
（ヒトヘルペスウイルス6型（HHV-６）及び7型（HHV-7））
　HHV-6は1986年、HHV-7は1990年に発見された２本鎖のDNAをゲノムとして持つDNAウイルスで、ヘルペスウイルス科Ｂ亜種に属する。
HHV-6にはHHV-6AとHHV-6Bの２種類（現在は亜種ではなく別個のウイルスとみなされている）があり、２種は塩基配列で約90%の相同性を有し、ヒト染色体にあるテロメアの繰り返し配列（[TAAGGG]n）と似た繰り返し配列（[TAACCC]n）をそのゲノムの両端に持つ（参考文献２５，２６）。
　殆んどの人は乳幼児期にHHV-6（A及びB）やHHV-7に初感染するが、HHV-6A及びHHV-6Bは潜伏感染先としてCNS組織（脳グリア細胞や海馬・神経細胞など）を選好し（参考文献２７）、また、HHV-7は唾液腺や嗅球に潜伏あるいは感染可能である。
　上記各ウイルスが侵入するホスト細胞の主な受容体としてHHV-6AはCD46（ほとんどの細胞で発現）、HHV-6BはCD46の他にCD134（CD4陽性及び／又はCD8陽性T細胞に発現）、HHV-7はCD4やCD8（活性化T細胞などで発現）が知られている（参考文献２５，２６）。 (Human herpesvirus type 6 (HHV-6) and type 7 (HHV-7))
HHV-6 is a DNA virus having double-stranded DNA as its genome, which was discovered in 1986 and HHV-7 in 1990, and belongs to the herpesvirus family B subspecies.
There are two types of HHV-6, HHV-6A and HHV-6B (currently considered as separate viruses, not subspecies), and the two types have about 90% homology in their nucleotide sequences, It has a repeat sequence ([TAACCC]n) similar to the telomere repeat sequence ([TAAGGG]n) on the human chromosome at both ends of its genome (References 25 and 26).
Most people are first infected with HHV-6 (A and B) and HHV-7 in infancy, but HHV-6A and HHV-6B are latent infection destinations of CNS tissue (such as brain glial cells and hippocampus/neuron). (Reference document 27), and HHV-7 can latently or infect salivary glands and olfactory bulb.
HHV-6A CD46 (expressed in most cells) as a main receptor of the host cells invaded by the above viruses, HHV-6B is CD134 (expressed in CD4 positive and/or CD8 positive T cells) in addition to CD46, HHV-7 is known to have CD4 and CD8 (expressed in activated T cells etc.) (references 25 and 26).
　初感染後、免疫機構によって排除されなかったHHV-6（A及びB）は宿主細胞に潜伏するが、その方法は独特で、ウイルスゲノムはヒト染色体のテロメアと一体となり潜伏する。また、宿主細胞の一染色体は一つのHHV-6（A又はB）ゲノムしか取り込まないこと、ウイルスゲノムが入り込む染色体としてX、1、6、7、9、10、11、12、17、18、19、22染色体が知られているが（参考文献２８）、これら以外の染色体中での潜伏は確認されていない。
　全人類の約1%がHHV-6A又はHHV-6Bのゲノムを遺伝によって引き継いでいるが、このウイルス（iciHHV-6A 及び iciHHV-6B）の病理作用はほとんど解明されていない。しかし、活性化のリスク要因になると指摘されている。
　参考文献２５：H Agut et al., Clinical Microbiology Reviews 28 (2): 313-335, 2015
　参考文献２６：湯華民他、「ウイルス」60 (2): 221-236, 2010
　参考文献２７：T Yoshikawa et al., Arch. Dis. Child. 83: 170-171, 2000
　参考文献２８：DA Clark, Clin. Microbiol. Infec. Vol. 22: 333-339, 2016 After initial infection, HHV-6 (A and B), which was not eliminated by the immune system, is latent in host cells, but the method is unique and the viral genome is integrated with the telomere of the human chromosome. In addition, one chromosome of the host cell incorporates only one HHV-6 (A or B) genome, and X, 1, 6, 7, 9, 10, 11, 12, 17, 18, as the chromosome into which the viral genome enters. Chromosomes 19 and 22 are known (reference document 28), but latency in other chromosomes has not been confirmed.
About 1% of all human beings inherit the HHV-6A or HHV-6B genome by inheritance, but the pathological effects of this virus (iciHHV-6A and iciHHV-6B) are poorly understood. However, it has been pointed out that it becomes a risk factor for activation.
Reference 25: H Agut et al., Clinical Microbiology Reviews 28 (2): 313-335, 2015.
Reference 26: Yukamin et al., "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もHHV-6（A及びB）と同一の繰り返し塩基配列（[TAACCC]n）をゲノムの両端に持ち、HHV-6脳炎類似の疾患の発症に関与するが（参考文献２９）、HHV-6（A及びB）のようにヒト染色体のテロメアと一体となり潜伏感染するか否かは解明されていない。
　HHV-7感染がもたらす疾患として、乳幼児期でのHHV-7初感染後に稀に惹起される突発性発疹や慢性疲労症候群などが知られている（参考文献２６）。しかし、HHV-6（A及びB）と同様、該ウイルスも潜伏感染中は人体に害作用を及ぼすことはなく、再活性化による急激な増殖がない限り、該ウイルスが潜伏感染している人の多くは大過なく一生を終えると考えられている。
　参考文献２９：M Parra et al., Virology Journal, 14 (97) : 1-5, 2017 HHV-7 also has the same repeated nucleotide sequence ([TAACCC]n) as HHV-6 (A and B) at both ends of the genome, and is involved in the development of HHV-6 encephalitis-like disease (Reference 29), It has not been clarified whether HHV-6 (A and B) is latently infected with telomeres of human chromosomes.
As a disease caused by HHV-7 infection, an exanthema subitum, chronic fatigue syndrome, etc., which are rarely caused after the initial infection of HHV-7 in infancy, are known (reference document 26). However, like HHV-6 (A and B), the virus does not have a harmful effect on the human body during latent infection, and as long as there is no rapid proliferation due to reactivation, the virus is latently infected by humans. Many of them are believed to end their lives without a big deal.
Reference 29: M Parra et al., Virology Journal, 14 (97): 1-5, 2017
　HHV-6Bは、HHV-6脳炎だけでなく、乳幼児の特発性発疹や初感染時の脳炎の主たる原因ウイルスでもある（参考文献２５，２６）。また、臨床的に観察される病理作用を発症させるHHV-6は殆どHHV-6Bであるが、その原因の詳細は解明されていない。
　HHV-6AやHHV-7が再活性化した場合の病理は十分に解明されていないが、HHV-6AはHHV-6Bと同様に感染細胞をアポトーシスに導き（非特許文献１０，参考文献３０）、またHHV-6AとHHV-7とは共感染できる（参考文献３１）。他方、HHV-6A及びHHV-7は共感染によりNK細胞から逃れる仕組みを有する（非特許文献１１）。
　参考文献３０：B Gu et al., Virology Journal 8 (5) 5: 1-10, 2011
　参考文献３１：M Ihara et al., Microbiol. Immunol. 45 (3): 225-232, 2001 HHV-6B is not only HHV-6 encephalitis, but also a major causative virus of idiopathic rash in infants and encephalitis at initial infection (References 25 and 26). Most of HHV-6 that causes clinically observed pathological effects is HHV-6B, but the cause thereof has not been clarified.
The pathology when HHV-6A and HHV-7 are reactivated has not been fully elucidated, but HHV-6A leads infected cells to apoptosis similarly to HHV-6B (Non-patent document 10, reference document 30). Moreover, HHV-6A and HHV-7 can be co-infected (reference 31). On the other hand, HHV-6A and HHV-7 have a mechanism to escape from NK cells by co-infection (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脳炎とAD）
　HHV-6脳炎の初発症状の典型的な事例は、遠い過去の出来事は答えられるが、日々のイベントなどの短期記憶が思いだせない（短期記憶障害）、自分の部屋やトイレの場所が分からない、などの見当識障害（認知障害）で、早期ADの典型的な症状と近似している。
　また、初発症状の頻度として、国内での調査では、意識障害（62%）、失見当識（52%）、記憶障害（48%）、痙攣（32%）、感覚障害（22%）、自立神経障害（6.2%）が報告されている（参考文献３２）。好発時期は臓器移植後12～30日と報告されており（参考文献３３）、治療開始が遅れると重大な後遺症や致死的となる可能性も高い。
　HHV-6脳炎の患者の頭部MRI検査では、大脳辺縁部（側頭内側の海馬、偏桃体）の両側性の異常所見がみられるが（参考文献３４）。これもADでは典型的に観察される病理所見である。しかし、HHV-6（A及びB）やHHV-7感染がAD発症や悪化のリスクファクターとなっても、ADの直接的な原因として指摘する調査、研究報告は見当たらない。
　また、2018年6月、AD患者の脳内（海馬傍回等）ではHHV-6A及びHHV-7のDNA数がそうでない人の約2倍との調査報告が公表されたが（非特許文献１２）、ADとこれらのウイルスのDNA濃度／数との関係は解明されていない。
　参考文献３２：日本造血細胞移植学会「造血細胞移植ガイドライン、ウイルス感染症の予防と治療HHV-6」, 2018, http://www.jshct.com/uploads/files/guideline/01_03_03_hhv6/pdf
　参考文献３３：DM Zerr et al., Clinical Infectious Diseases. 2002; 34: 309-3017, 2002
　参考文献３４：T Noguchi et al., Am. J. Neuroradiol. 27: 2191-2195, 2006 (HHV-6 encephalitis and AD)
A typical case of the first symptoms of HHV-6 encephalitis can answer distant past events, but can not remember short-term memory such as daily events (short-term memory disorder), do not know the location of their room or toilet, Such disorientation (cognitive impairment) is similar to the typical symptoms of early AD.
As for the frequency of initial symptoms, in a domestic survey, consciousness disorder (62%), disorientation (52%), memory disorder (48%), convulsion (32%), sensory disorder (22%), independence. Neuropathy (6.2%) has been reported (Reference 32). It is reported that the frequency of occurrence is 12 to 30 days after organ transplantation (Ref. 33), and if the start of treatment is delayed, there is a high possibility of serious sequelae and fatalities.
MRI examination of the head of patients with HHV-6 encephalitis shows bilateral abnormal findings in the limbic region (medial temporal hippocampus, amygdala) (Reference 34). This is also a pathological finding typically observed in AD. However, even if HHV-6 (A and B) or HHV-7 infection is a risk factor for AD development or exacerbation, there is no research or report pointing out as a direct cause of AD.
Further, in June 2018, a research report was published that the number of HHV-6A and HHV-7 DNAs in the brains of AD patients (parahippocampal gyrus, etc.) was about twice that of those who did not (non-patent document 12), the relationship between AD and DNA concentration/number of these viruses has not been elucidated.
Reference 32: Japan Hematopoietic Cell Transplantation Society "Hematopoietic Cell Transplantation Guidelines, Prevention and Treatment of Viral Infections 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.
　これらの結果、３０年以上にわたり、様々な仮説に従って多数のAD治療薬が製薬企業や大学等の研究機関で研究され、開発されてきたが（非特許文献３、４、参考文献６、３５）、HHV-6（HHV-6A及び／又はHHV-6B）やHHV-7を標的としたAD治療薬の開発は何一つ知られておらず、また、AD患者での該ウイルスを標的とした治験が試みられたことはない。
　また、HHV-6（A及びB）やHHV-7に対する抗ウイルス効果を有する化合物やその医薬組成物（製剤）、例えばホスカルネット、ファムシクロビル、アシクロビル、バラシクロビル、ガンシクロビル、シドフォビルを含むフォスフォネート誘導体、抗CD40抗体、ピリドキノキサリン、ピラソロピリジン誘導体、フルオレセント化合物、SITH-1阻害剤、ナノ・エマルジョン組成物、グレムリン誘導体などの抗ウイルス剤の先行特許でADを適応疾患とするものは見当たらない。
　参考文献３５：Y Dong et al., International J. Molecular Sciences 20 (588): 1-24, 2019 As a result, over 30 years, many AD therapeutic agents have been studied and developed by research institutions such as pharmaceutical companies and universities according to various hypotheses (Non-patent Documents 3, 4 and References 6, 35). , The development of AD therapeutic drugs targeting HHV-6 (HHV-6A and/or HHV-6B) and HHV-7 is unknown, and clinical trials targeting the virus in AD patients have been It has never been tried.
In addition, compounds having an antiviral effect against HHV-6 (A and B) and HHV-7 and pharmaceutical compositions (formulations) thereof, such as foscarnet, famciclovir, acyclovir, valacyclovir, ganciclovir and phosphophos, including cidofovir. Anti-viral agents such as nate derivative, anti-CD40 antibody, pyridoquinoxaline, pyrazolopyridine derivative, fluorescent compound, SITH-1 inhibitor, nano-emulsion composition, gremlin derivative, etc. I can't find anything.
Reference 35: Y Dong et al., International J. Molecular Sciences 20 (588): 1-24, 2019
　HHV-6脳炎に対する適応症を得ている医薬品（抗ヘルペスウイルス剤を含む）は、世界的に見ても存在しなかったが、臨床現場では、十数年にわたり主にホスカルネット及びガンシクロビルの製剤が適応外で治療に用いられてきた（非特許文献１３、参考文献２０、３２）。
　これらの実績をもとにして、2018年2月、日本造血細胞移植学会が定めた「造血細胞移植ガイドライン」（参考文献３２）は、「HHV-6脳炎に対する治療の基本は、すみやかにホスカルネットのフルドーズ（60mg/kg、8時間ごとに1日3回）での投与を開始すること、治療期間は最低3週間」であると定めている。 Although there are no drugs (including anti-herpesviruses) that have been indicated for HHV-6 encephalitis in the world, clinical trials have focused on foscarnet and ganciclovir for over a decade. The formulation has been used for therapy off label (Non-patent Document 13, References 20, 32).
Based on these achievements, the “Hematopoietic Cell Transplantation Guidelines” (Reference 32) established by the Japan Society for Hematopoietic Cell Transplantation in February 2018, states that “The basics of treatment for HHV-6 encephalitis are promptly Hoscal It is prescribed that the administration of net full dose (60 mg/kg, 3 times a day every 8 hours) should be started, and the treatment period should be at least 3 weeks.
　アルツハイマー病（「AD」）の発症原因として様々な仮説が提唱され、その仮説に依拠したAD治療薬の開発が日米欧で数十年にわたり行われてきた。しかし、最も期待されていたAβを標的とした新薬開発でも、実用化に至った化合物はない。ADを予防し、進行を止め、改善を図れる根本的なAD治療薬の開発が切望されている。 Various hypotheses have been proposed as the cause of the onset of Alzheimer's disease (“AD”), and the development of AD therapeutic drugs based on that hypothesis has been carried out for decades in Japan, the United States, and Europe. However, even with the most anticipated new drug development targeting Aβ, no compound has been put to practical use. There is a strong demand for the development of a fundamental AD therapeutic drug that can prevent AD, stop its progression, and improve it.
　本発明者は、認知症の発症に潜伏ウイルスであるHSV-1の感染歴が関与しているという点に注目し、通常は感染症状を示さない脳内潜伏ウイルスを標的とすることによるADの治療を考えた。
　即ち、脳内のグリア細胞や神経細胞等に潜伏感染しているが、かかる状況下では感染に伴う炎症症状（発熱、発赤等）を示さない潜伏ウイルスを標的とし、その再活性化を抗ウイルス剤で抑制することによるAD発症の予防及び治療である。抗ウイルス剤をADの治療及び予防に用いる根拠は、脳内での潜伏ウイルスの再活性化が自然免疫応としてのAβの産生を促し、また、活性化ウイルスの感染がもたらす病理（炎症や細胞死など）がADの発症と悪化に深く関与しているからである。 The present inventor has noted that the history of infection with the latent virus, HSV-1, is involved in the development of dementia, and AD of AD by targeting a brain latent virus that does not normally show infectious symptoms. I thought about treatment.
That is, although the latent infection of glial cells and nerve cells in the brain is carried out, in such a situation, the latent virus that does not show the inflammatory symptoms (fever, redness, etc.) associated with the infection is targeted, and its reactivation is carried out by antivirus. It is the prevention and treatment of AD onset by suppressing with a drug. The basis for using antiviral agents for the treatment and prevention of AD is that reactivation of latent virus in the brain promotes the production of Aβ as an innate immune response, and the pathology (inflammation and cell Death) is deeply involved in the onset and exacerbation of AD.
　ウイルスの潜伏感染やその再活性化は、多種ウイルスの共同作用によって成り立っているとの報告がある（非特許文献１５）。発明者は、この点に注目し、AD患者の脳内、特に海馬で、脳内での潜伏感染が知られているHHV-6A、HHV-6B及びHHV-7（以下、「HHV-6/7」という）の共同作用で再活性化の際の感染症状が悪化しあるいは顕在化が起きる可能性が高いと考えた。
　Aβの産生は、脳内に現れたウイルスへの自然免疫応答と考えれば、HHV-6/7が再活性化した場合、神経細胞が産生、放出したAβがこれらウイルスの表面にとりついて動きを止め、脳内でマクロファージの役をするミクログリアによるウイルスの貪食を助ける。また、ミクログリアの貪食を逃れたウイルスは単独であるいはAβとの複合体となりエンドサイトーシスによって神経細胞内に取り込まれ、蓄積されるのではないか。
　上記仮説が正しければ、ADにおける神経細胞内におけるAβ蓄積の機構が説明できる。
　つまり、AD患者での神経細胞内のAβ蓄積には、少なくとも脳内で潜伏感染していたHHV-6/7の再活性化に伴う感染が関与しており、この再活性化・感染を抑制すれば、Aβが関与するADの発症（図１）を予防しあるいは治療が可能となる。
　Aβ産生酵素（βセクレターゼなど）の作用を阻害してAβの産生のみを抑えた場合、ウイルスに対する防御力が低下し、再活性化したウイルスの増殖を許してしまう可能性がある。
　発明者は、この脳内での潜伏ウイルス、特にHHV-6/7の再活性化がAβ産生の根本原因であるとすれば、かかる潜伏ウイルスの排除（具体的には再活性化したときの感染及びウイルス増殖の抑制）が、Aβの産生及びそのオリゴマー化の抑止、神経細胞内でのAβ（オリゴマー）の蓄積及びそれに起因するCa＋＋濃度の急激な上昇など神経細胞死をもたらす様々な要因を抑制し、ADに対する根本的な治療薬となる可能性があると考えた（図２）。 It has been reported that latent infection of virus and its reactivation are based on the synergistic action of various viruses (Non-Patent Document 15). The inventor pays attention to this point, in the brain of AD patients, particularly in the hippocampus, HHV-6A, HHV-6B and HHV-7 known to have latent infection in the brain (hereinafter, referred to as "HHV-6/ It is highly likely that the synergistic effect of "7") will worsen or manifest infection symptoms upon reactivation.
Considering that Aβ production is an innate immune response to the virus that appears in the brain, when HHV-6/7 is reactivated, Aβ produced and released by nerve cells attaches to the surface of these viruses and stops moving. , Helps phagocytosis of viruses by microglia, which act as macrophages in the brain. In addition, the virus that escapes phagocytosis of microglia may be taken up and accumulated in nerve cells by endocytosis alone or as a complex with Aβ.
If the above hypothesis is correct, the mechanism of Aβ accumulation in neurons in AD can be explained.
In other words, Aβ accumulation in nerve cells in AD patients involves at least infection associated with reactivation of HHV-6/7 that was latently infected in the brain, and suppresses this reactivation/infection. Then, it becomes possible to prevent or treat the onset of AD (FIG. 1) associated with Aβ.
If only the production of Aβ is suppressed by inhibiting the action of Aβ-producing enzyme (β-secretase etc.), the defense power against the virus may be lowered and the reactivated virus may be allowed to grow.
The inventor, if the reactivation of this latent virus in the brain, especially HHV-6/7 is the root cause of Aβ production, the elimination of such latent virus (specifically, when reactivating Inhibition of Aβ production and oligomerization, inhibition of Aβ production and its oligomerization, accumulation of Aβ (oligomer) in nerve cells, and the resulting rapid increase of Ca ++ concentration resulting in various neuronal cell death It was thought that it could be a fundamental therapeutic drug for AD (Fig. 2).
　そこで、上記仮説を検証すべく、ヒトの中枢神経系内でHHV-6（A及びB）及び／又はHHV-7（以下、「HHV-6/7」ともいう）が潜在的に感染した状態の神経細胞を模したモデル細胞として、HHV-6/7感染させた培養ヒト神経細胞株を用いる以下の実験系を構築した。尚、動物モデルでの試験系を構築しなかったのは、HHV-6/7がヒト特異的なヘルペスウイルスである為、適切な感染動物モデルが得られないからである。
１）HHV-6A,HHV-6B及びHHV-7の各ウイルス溶液の調製
２）Aβのオリゴマーの調製
　試験に用いるAβとして、老人斑の主構成成分であるAβオリゴマーの主な組成分子であるアミノ酸42個で構成される可溶性のAβ42を選択した。
３）被験細胞
　ヒト由来神経細胞株として標準的に使用されているSH-SY5Y株（ヒト由来神経芽細胞株）を選択
４）試験サンプル群
　試験には以下のサンプル群を構築し、6濃度（0, 0.1, 1.0, 10, 100, 1000 nM）のAβの添加群について被験細胞を24時間及び48時間培養して比較した。尚、各ウイルスは実施例１の過程で調整したウイルス溶液である。また、ホスカルネットとは同成分を唯一の有効成分とする点滴静注用製剤「ホスカビル（登録商標）」である。 Therefore, in order to verify the above hypothesis, HHV-6 (A and B) and/or HHV-7 (hereinafter, also referred to as “HHV-6/7”) is potentially infected in the human central nervous system. The following experimental system was constructed using a cultured human nerve cell line infected with HHV-6/7 as a model cell simulating the nerve cell of. In addition, the reason why the test system in the animal model was not constructed is that since HHV-6/7 is a human-specific herpesvirus, an appropriate infected animal model cannot be obtained.
1) Preparation of HHV-6A, HHV-6B, and HHV-7 virus solutions 2) Preparation of Aβ oligomer Amino acid, which is the main constituent molecule of Aβ oligomer, which is the main constituent of senile plaques Soluble Aβ 42 consisting of 42 was selected.
3) Test cells Select SH-SY5Y strain (human-derived neuroblast cell line) that is normally used as a human-derived neuronal cell line 4) Test sample group For the test, the following sample groups were constructed and 6 concentrations ( (0, 0.1, 1.0, 10, 100, 1000 nM) Aβ-added groups were compared with each other by culturing the test cells for 24 hours and 48 hours. Each virus is a virus solution prepared in the process of Example 1. In addition, foscarnet is a foscavir (registered trademark) formulation for intravenous drip infusion containing the same ingredient as the only active ingredient.
Figure JPOXMLDOC01-appb-T000003
 ５）試験方法の概要
　被験細胞を培養中の培地にHHV-6/7の各ウイルス溶液と複数濃度のAβオリゴマーを添加し、HHV-6/7感染細胞が細胞内に「ウイルス複合体」してAβを取り込み、細胞内でAβを過剰に蓄積するか否かを調べる。更に、細胞外の外のAβ及び細胞内に取り込まれたAβが細胞毒（増殖の抑制、細胞死など）を有するか否かを検証した。
Figure JPOXMLDOC01-appb-T000003
 5) Outline of the test method Each virus solution of HHV-6/7 and multiple concentrations of Aβ oligomers were added to the culture medium of the test cells, and the HHV-6/7 infected cells formed "virus complex" in the cells. Uptake Aβ, and examine whether Aβ excessively accumulates in cells. Furthermore, it was verified whether extracellular Aβ outside and Aβ taken into the cell have cytotoxicity (suppression of proliferation, cell death, etc.).
　試験の結果、細胞外にウイルス（HHV-6/7）がいるか否かに拘わらず、培地（細胞外）内のAβ42は被験細胞に取り込まれることが示された。ただし、Aβの添加濃度0.1～10nMにおいて、Aβ42の細胞内濃度はHHV-6AとHHV-7の混合液、HHV-6BとHHV-7の混合液、HHV-7単独のウイルス溶液添加群では減少傾向を示したが、ウイルス溶液未添加群ではそのような減少傾向はみられなかった（図７、８）。このことは細胞外のウイルスがAβ42と結合し、細胞内へのAβ42の移行を抑制することを示唆した。
　反面、試験条件下においてHHV-6/7ウイルス溶液の添加は細胞内Aβの増加を示さなかった。
　また、感染した被験細胞内でのウイルス（HHV-6）DNA数を調べた試験では、Aβ42の添加濃度に比例してDNA数は減少したが、Aβ42の未添加群ではそのような減少は観察されなかった（図８）。このことは、細胞外Aβは、細胞外に存在するウイルスの細胞内への取り込みを抑制していることを示唆した。
　以上の試験結果は、Aβ42のウイルス感染に対する防御作用（細胞内へのウイルスの取り込み抑制）はHHV-6/7に対しても有すること示した。しかし、「ウイルスAβ複合体」（ウイルスと結合したAβ42）が細胞内に取り込まれ、細胞内でのAβ42蓄積をもたらすことは、試験条件下では検証できなかった。 As a result of the test, it was shown that Aβ 42 in the medium (extracellular) was taken up by the test cells regardless of whether the virus (HHV-6/7) was extracellular. However, when the concentration of Aβ added was 0.1 to 10 nM, the intracellular concentration of Aβ 42 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 was added. Although a decreasing tendency was shown, such a decreasing tendency was not observed in the virus solution-free group (FIGS. 7 and 8). This suggested that the extracellular virus binds to Aβ 42 and suppresses Aβ 42 translocation into the cell.
On the other hand, addition of HHV-6/7 virus solution under the test conditions did not show an increase in intracellular Aβ.
Also, infected with the virus (HHV-6) study examined the number DNA in a subject within the cell, the number of DNA in proportion to the concentration of added A [beta] 42 has been reduced, such reduction in the non-addition group of A [beta] 42 Was not observed (Fig. 8). This suggested that extracellular Aβ suppressed the intracellular uptake of the virus existing outside the cell.
The above test results showed that Aβ 42 also has a protective action against viral infection (suppression of viral uptake into cells) against HHV-6/7. However, it was not possible to verify under the test conditions that the "viral Aβ complex" (Aβ 42 bound to the virus) was taken up into the cell, resulting in intracellular Aβ 42 accumulation.
　これまでHHV-6/7に感染した細胞はアポトーシスするとされていた（R Ichimi et al., Journal of Medical Virology, 58 (1): 63-68, 1999、及び非特許文献１０、１１、参考文献３０）ことから、上記試験に関連して各ウイルスによる感染細胞死の有無を検証した（実施例５）。
　HHV-6Aに関しては、未感染のHSB-2株（ヒトTリンパ球芽細胞株）、HHV-6B及びHHV-7に関して未感染のSup-T1株（ヒトTリンパ球由来T細胞株）に（実施例１）で得られた各ウイルス溶液を添加し、ウイルス感染の有無、感染細胞の形態の変化を観察した（実施例５）。
　その結果、HHV-6Aの未感染細胞群（図１０－１）では、各細胞の形や大きさは均一で、死亡した細胞は殆ど観察されていない。しかし、感染細胞群（図１０－２）では、肥大した細胞及び死細胞が散見された。
　また、HHV-6B及びHHV-7に関しては、未感染のSup-T1株群（図１１－１）では、死細胞は見られず、細胞の形、大きさも均一である。しかし、HHV-6B感染細胞群（図１１－２）及びHHV-7感染細胞群（図１２）で僅かな死細胞の他に、細胞表面がゴツゴツした細胞、肥大した細胞が散見された。 Until now, cells infected with HHV-6/7 were said to be apoptotic (R Ichimi et al., Journal of Medical Virology, 58 (1): 63-68, 1999, and Non-Patent Documents 10, 11, References) Therefore, the presence or absence of infected cell death by each virus was verified in connection with the above test (Example 5).
For HHV-6A, uninfected HSB-2 strain (human T lymphocyte blast cell line), and for HHV-6B and HHV-7 uninfected Sup-T1 strain (human T lymphocyte-derived T cell line) ( Each virus solution obtained in Example 1) was added, and the presence or absence of virus infection and changes in the morphology of infected cells were observed (Example 5).
As a result, in the HHV-6A uninfected cell group (FIG. 10-1), the shape and size of each cell were uniform, and almost no dead cells were observed. However, in the infected cell group (FIG. 10-2), enlarged cells and dead cells were scattered.
Regarding HHV-6B and HHV-7, dead cells were not seen in the uninfected Sup-T1 strain group (FIG. 11-1), and the cell shape and size were uniform. However, in the HHV-6B-infected cell group (FIG. 11-2) and the HHV-7-infected cell group (FIG. 12), in addition to a few dead cells, cells with rugged cell surfaces and enlarged cells were observed.
　本発明者は上記試験結果から、HHV-6/7に感染したT細胞等がアポトーシスの他にネクローシスをも惹起し、この異なる二種の細胞死、特にネクローシスがもたらす病理に、上記ウイルスの再活性化に対するAβの病理（選択的な神経機能の病変・細胞死、Ca++恒常性の阻害、シナプス損傷、ミクログリアの活性化やアストロサイトの反応促進、インスリン抵抗性の増加等：参考文献３）との相乗作用がAD発症の根本的な原因であることを見出した。
　即ち、加齢下において、海馬で生じたHHV-6/７の再活性化に伴う軽微な炎症（HHV-6脳炎）への応答として神経細胞からのAβの産生の他に、脆弱化したBBBを通過して当該部位に活性化T細胞が遊走する。このとき、ウイルス感染した一部のT細胞がネクローシスし、死亡細胞から放出されたHMGB-1のようなDAMPsや感染した細胞（T細胞に限定されない）が発現するCCL2のようなケモカインの作用で、細胞障害性T細胞やNK細胞等の免疫関連細胞の感染部位への遊走・凝集が増強する。この結果、AD患者では再活性化ウイルスに感染した部位（特に海馬）で局所的な細胞死が持続的に進行し、再生能を超えるほどの神経細胞死が亢進すると不可逆的に神経機能が傷害を受ける、との仮説（「HHV-6脳炎カスケード仮説」）が成立する。
　上記により、HHV-6（A及びB）及びHHV-7に対する優れた効果を有するマルチヘルペスウイルス剤によって、ADの進行を止めるだけでなくその根本的な治療が短期間（３～６ヵ月）で実現できる可能性があることを見出し、本発明を完成した。 From the above test results, the present inventors have shown that HHV-6/7-infected T cells and the like also induce necrosis in addition to apoptosis, and the pathology caused by these two different types of cell death, particularly necrosis, causes Pathology of Aβ for activation (selective lesions/cell death of nerve function, inhibition of Ca ++ homeostasis, synapse damage, activation of microglia and astrocyte reaction, increase of insulin resistance, etc.: Reference 3 ) Is a fundamental cause of AD onset.
That is, under aging, in addition to Aβ production from nerve cells in response to slight inflammation (HHV-6 encephalitis) associated with reactivation of HHV-6/7 that occurred in the hippocampus, weakened BBB The activated T cells migrate to the relevant site through the cell. At this time, some virus-infected T cells undergo necrosis, and DAMPs such as HMGB-1 released from dead cells and chemokines such as CCL2 expressed by infected cells (not limited to T cells) are acted upon. , Migration and aggregation of immune-related cells such as cytotoxic T cells and NK cells to the site of infection are enhanced. As a result, in AD patients, local cell death continuously progressed at the site (especially hippocampus) infected with reactivating virus, and nerve function was irreversibly impaired if nerve cell death increased beyond the regenerative capacity. The hypothesis of being received ("HHV-6 encephalitis cascade hypothesis") is established.
Based on the above, the multi-herpesvirus agent, which has an excellent effect on HHV-6 (A and B) and HHV-7, not only stops the progression of AD but also its fundamental treatment in a short period (3 to 6 months). The present invention has been completed by finding out that it can be realized.
　本発明において、ADは以下のプロセスにより発症し悪化すると考える（「HHV-6脳炎カスケード仮説」）。
１）殆どの人は幼少時にHHV-6（A及びB）及びHHV-7に感染するが、該ウイルス、特にHHV-6（A及びB）はCNS組織（脳のグリア細胞、特にアストロサイト及び再生可能な神経細胞を持つ海馬や嗅球など）に潜伏している（JM Reynaud et al.,ISRN Virology 2013, vol.2013: 1-11, 2013）。
２）ヒト細胞では上記ウイルスは単球やCD４やCD8陽性のT細胞などの免疫細胞に感染・潜伏するだけでなく、これらの細胞の中で増殖できる（参考文献３４）。従って、再活性化に伴って感染細胞が生成するCCL2などのケモカインによって該ウイルスの主な受容体となるCD46（HHV-6A）、CD134（HHV-6B）、CD4（HHV-7）を発現する活性化T細胞などが感染部位に遊走する場合、貪食ではなく、反って該ウイルスの増殖をもたらす。
３）海馬では、歯状回にある神経幹細胞が前駆神経細胞、顆粒細胞、錐体細胞へと分化することで神経細胞数の恒常性を維持している。
４）潜伏DNAウイルスは宿主細胞の分化や分裂の際に再活性化することから、海馬の神経幹細胞や前駆神経細胞に潜伏しているDNAウイルスのHHV-6は再活性化する可能性が高い。
５）潜伏HHV-6の再活性化は、感染に伴う軽度の炎症（HHV-6脳炎）を近傍に惹起する。このウイルス感染を察知した神経細胞が自然免疫応答としてAβを産生・分泌し、これがウイルスを捕捉し、あるいはアストロサイトが感知し、脳内でマクロファージの役を担っているミクログリアに伝え、その処理に委ねる。この時、TNF-αやIFN等の炎症性サイトカインやCCL2等のケモカインの発現状況によってはT細胞が活性化され、BBBを通過して感染個所に遊走する。
６）HHV-6の潜伏方法から一細胞内で潜伏できるウイルスの数が少ない。従って、再活性化するウイルスの数も少なく、通常は、脳内でマクロファージの役を担うミクログリアやT細胞によって貪食される。この結果、HHV-6の再活性化に伴うウイルス感染は、例外的な場合を除き、病的な臨床症状（発熱等）を惹起することはない。
　HHV-6/7に起因するウイルス性の脳炎が稀にしか発症しないことは、このことを示唆している。しかし、ウイルス再活性化の残渣として該ウイルスDNAを含むAβプラーク（老人斑）が脳内の感染部位に散在することはありえる。AD患者において発症の15年以上も前から脳内にAβプラーク（老人斑）の蓄積がみられるが、上記により説明が可能となる。
７）加齢によって最初に脆弱化するBBBは海馬BBBである（A Montagne et al., Neuron, 85 (2):296-302, 2015)。従って、海馬でHMGB-1などのDAMPsによる炎症が増強している場合、CCL2などのケモカインの誘引によって末梢血中のNK細胞や活性化T細胞が脆弱化したBBBを通過して脳の炎症部位に遊走する。特にT細胞は比較的容易にBBBを通過できるため（松井真、臨床神経53(11):898-900、2013）、活性化T細胞の感染部位への遊走が亢進する。
８）この結果、CD4やCD8を発現した活性化T細胞内で増殖できるHHV-6（A及びB）はその数を増やし、感染を拡大、重篤化させる。更には、生体内でウイルス等の病原体に対する主な攻撃役であるNK細胞の遊走を招く結果、NK細胞や障害性T細胞（CD8が発現）による細胞死（多くはアポトーシス）が増強する。
９）つまり、再活性化HHV-6（A及び／又はB)の感染が起こす炎症→活性化Ｔ細胞の遊走→Ｔ細胞への感染→感染Ｔ細胞ネクローシス→DAMPs放出→炎症の増強・感染細胞（神経細胞を含む）アポトーシス／ネクローシス→DAMPs放出→活性化T細胞・NK細胞の感染部位への遊走・浸潤の増強→更なる細胞死、という負のサイクルが、感染部位での神経機能に関わる細胞（アストロサイト及び神経細胞を含む）死を亢進させる。
　特に嗅球などに潜伏感染していたHHV-7が再活性化して海馬に移動、あるいは海馬内で再活性化した場合、HHV-6Aとの共感染により感染細胞が周辺の未感染神経細胞にTRAILを発現させてNK細胞による細胞死を拡大させる。これらの結果、ウイルス感染に対するAβの作用と相まって神経幹細胞や前駆細胞を有する組織（海馬及び大脳辺縁系）では、HHV-6（A及びB）再活性化に伴う脳炎（HHV-6脳炎）等の病理が顕在化し、再生能を超える細胞死が緩徐にかつ確実に進み、神経機能が徐々に損なわれADが発症する。 In the present invention, AD is considered to be developed and exacerbated by the following processes (“HHV-6 encephalitis cascade hypothesis”).
1) Most humans are infected with HHV-6 (A and B) and HHV-7 at an early age, and the virus, especially HHV-6 (A and B), is infected with CNS tissues (glial cells in the brain, especially astrocytes and It is latent in the hippocampus and olfactory bulb with reproducible nerve cells (JM Reynaud et al., ISRN Virology 2013, vol.2013: 1-11, 2013).
2) In human cells, the virus not only infects and latencies immune cells such as monocytes and CD4 and CD8 positive T cells, but also can proliferate in these cells (Reference 34). Therefore, chemokines such as CCL2 produced by infected cells upon reactivation express CD46 (HHV-6A), CD134 (HHV-6B), and CD4 (HHV-7), which are the main receptors of the virus. When activated T cells and the like migrate to the site of infection, they are not phagocytosed but warp and cause the growth of the virus.
3) In the hippocampus, neural stem cells in the dentate gyrus maintain the homeostasis of the number of neurons by differentiating into progenitor cells, granule cells and cone cells.
4) Since latent DNA viruses are reactivated during host cell differentiation and division, HHV-6, which is a DNA virus latent in hippocampal neural stem cells and progenitor neurons, is likely to be reactivated. ..
5) Reactivation of latent HHV-6 causes mild inflammation (HHV-6 encephalitis) associated with infection in the vicinity. Neurons that detect this viral infection produce and secrete Aβ as an innate immune response, which captures the virus or is detected by astrocytes and transmitted to microglia, which plays the role of macrophages in the brain, and processes it. Entrust. At this time, depending on the expression status of inflammatory cytokines such as TNF-α and IFN and chemokines such as CCL2, T cells are activated and migrate through the BBB to the infected site.
6) Due to the HHV-6 latency method, the number of viruses that can be latent in one cell is small. Therefore, the number of viruses that reactivate is small, and they are usually phagocytosed by microglia and T cells that play a role of macrophages in the brain. As a result, viral infection associated with reactivation of HHV-6 does not cause pathological clinical symptoms (fever, etc.) except in exceptional cases.
The rare occurrence of viral encephalitis due to HHV-6/7 suggests this. However, it is possible that Aβ plaques (senile plaques) containing the viral DNA as a residue of virus reactivation are scattered at the infection site in the brain. Accumulation of Aβ plaques (senile plaques) in the brain has been seen in AD patients for more than 15 years before the onset of the disease, which can be explained by the above.
7) The BBB that is first vulnerable to aging is the hippocampal BBB (A Montagne et al., Neuron, 85 (2):296-302, 2015). Therefore, when inflammation due to DAMPs such as HMGB-1 is enhanced in the hippocampus, NK cells and activated T cells in peripheral blood pass through the weakened BBB by the attraction of chemokines such as CCL2 and the inflammatory site of the brain To run to. In particular, since T cells can cross the BBB relatively easily (Matsui Makoto, Clinical Neuron 53(11):898-900, 2013), migration of activated T cells to the infected site is enhanced.
8) As a result, HHV-6 (A and B), which can proliferate in activated T cells expressing CD4 and CD8, increases its number and spreads and seriously infects the infection. Furthermore, as a result of migration of NK cells, which is a major attacker against pathogens such as viruses, in vivo, cell death (mostly apoptosis) due to NK cells and impaired T cells (expressing CD8) is enhanced.
9) In other words, inflammation caused by infection with reactivated HHV-6 (A and/or B)→migration of activated T cells→infection of T cells→infected T cell necrosis→release of DAMPs→enhancement of inflammation/infected cells Apoptosis/necrosis (including nerve cells)→DAMPs release→migration of activated T cells/NK cells to the infected site/enhancement of invasion→further cell death, the negative cycle is involved in neural function at the infected site Promotes cell (including astrocyte and nerve cell) death.
In particular, when HHV-7 that had been latently infected with the olfactory bulb, etc. is reactivated and migrates to the hippocampus, or is reactivated in the hippocampus, the infected cells TRAIL to the surrounding uninfected nerve cells by co-infection with HHV-6A. To expand cell death by NK cells. As a result, encephalitis (HHV-6 encephalitis) associated with HHV-6 (A and B) reactivation was found in tissues (hippocampus and limbic system) that have neural stem cells and progenitor cells coupled with the action of Aβ on viral infection. Pathology such as is manifested, cell death exceeding regenerative ability progresses slowly and surely, nerve function is gradually impaired, and AD develops.
「HHV-6脳炎カスケード仮説」におけるAD発症に至るプロセスの概要は、（図１）のとおりである。 The outline of the process leading to AD onset in the "HHV-6 encephalitis cascade hypothesis" is as shown in (Fig. 1).
　以上から、本発明は以下の発明を包含する。
［１］アルツハイマー病（「AD」）を治療または予防する為の医薬組成物であって、ヒトヘルペスウイルス6型A及びB（以下併せて「HHV-6」という）及び／又は7型（「HHV-7」）に対する抗ウイルス活性を持つ化合物を有効成分とし、薬学的に許容される担体を含む医薬組成物及びその使用。
　アルツハイマー病（「AD」）を治療（予防を含む）する為の、「HHV-6」及び／又は「HHV-7」に対する抗ウイルス活性を持つ有効量の化合物は、更に同等の有効量で、脳炎などの中枢神経疾患を惹起する単純ヘルペスウイルス1型（「HSV-1」）及びサイトメガロウイルス（「CMV」）などに対しても活性を有するマルチ抗ウイルス作用を持つ化合物であることが望ましい。
［２］前記化合物が、更にHSV-1及びCMVに対する抗ウイルス活性を有する化合物である、前記［１］に記載の医薬組成物及びその使用。
　ここで、本発明の医薬組成物は、「HHV-6」及び「HHV-7」のみならず、脳炎などの中枢神経疾患を惹起するHSV-1及びCMVなどに対しても同等又は近似の有効量で活性を有する、すなわちマルチ抗ウイルス作用を有する化合物を有効成分とする。
［３］前記化合物が、標的ウイルス由来のDNAポリメラーゼのピロリン酸結合部位に結合し、選択的にウイルスの増殖を阻害する化合物である、前記［１］又は［２］に記載の医薬組成物及びその使用。
［４］前記化合物が、ホスホノ酢酸もしくはホスホノギ酸又はそれらの誘導体であるピロリン酸アナログである、前記［１］～［３］のいずれかに記載の医薬組成物及びその使用。
　なお、ホスホノ酢酸又はその誘導体は、ホスホノギ酸又はそれらの誘導体と同様に抗ウイルス活性を有することが知られている。
［５］前記化合物が、下記基本構造式（式１）で示されるホスホノギ酸もしくはその塩、又はこれらの溶媒和物（これらを以下「ホスホノギ酸誘導体」という）である、前記［１］～［４］のいずれかに記載の医薬組成物及びその使用。 From the above, the present invention includes the following inventions.
[1] A pharmaceutical composition for treating or preventing Alzheimer's disease (“AD”), which comprises human herpesvirus type 6 A and B (hereinafter collectively referred to as “HHV-6”) and/or type 7 (“ A pharmaceutical composition comprising a compound having antiviral activity against HHV-7") as an active ingredient and a pharmaceutically acceptable carrier, and use thereof.
An effective amount of a compound having antiviral activity against “HHV-6” and/or “HHV-7” for treating (including prevention) Alzheimer's disease (“AD”) is a further equivalent effective amount, It is desirable that the compound has a multi-antiviral action that is active against herpes simplex virus type 1 (“HSV-1”) and cytomegalovirus (“CMV”) that cause central nervous system diseases such as encephalitis ..
[2] The pharmaceutical composition and the use thereof according to [1] above, wherein the compound is a compound having antiviral activity against HSV-1 and CMV.
Here, the pharmaceutical composition of the present invention is not only “HHV-6” and “HHV-7”, but also equivalent or similar effective to HSV-1 and CMV which cause central nervous diseases such as encephalitis. A compound having an active amount, that is, having a multi-antiviral effect is used as an active ingredient.
[3] The pharmaceutical composition according to the above [1] or [2], wherein the compound is a compound that binds to the pyrophosphate binding site of the DNA polymerase derived from the target virus and selectively inhibits the growth of the virus. Its use.
[4] The pharmaceutical composition according to any one of [1] to [3] above, and the use thereof, wherein the compound is a phosphonoacetic acid, a phosphonoformic acid, or a pyrophosphate analog which is a derivative thereof.
It is known that phosphonoacetic acid or its derivative has antiviral activity similarly to phosphonoformic acid or its derivative.
[5] The above-mentioned [1] to [1], wherein the compound is phosphonoformic acid represented by the following basic structural formula (Formula 1) or a salt thereof, or a solvate thereof (hereinafter, referred to as “phosphonoformic acid derivative”). [4] The pharmaceutical composition according to any one of [4] and the use thereof.
Figure JPOXMLDOC01-appb-C000004
 　なお、（式１）の「ホスホノギ酸誘導体」のうち最も好ましい化合物は、下記（式２）で表されるホスギノ酸３ナトリウムの６水和物（以下「ホスカルネット」という）であり、（JNN）／INN表記でFoscarnet Sodium と表記される。
Figure JPOXMLDOC01-appb-C000004
 The most preferable compound among the "phosphonoformic acid derivatives" of (Formula 1) is trisodium phosphinate hexahydrate (hereinafter referred to as "foscarnet") represented by the following (Formula 2), JNN)/INN notation is written as Foscarnet Sodium.
Figure JPOXMLDOC01-appb-C000005
 （式２）
Figure JPOXMLDOC01-appb-C000005
 (Formula 2)
［６］前記医薬組成物がホスカルネットを有効成分とする注射剤からなり、ホスカルネットの有効量として60～180mg/kg/日を、１日２回又は３回に分けて、髄腔内投与あるいは点滴静注により３日ないし１０日間継続して投与することによる先制療法のための医薬組成物である、前記［５］に記載の医薬組成物及びその使用が例示できる。
　ここで、本発明のホスカルネットかを有効成分とする注射剤をADの先制療法として用いる場合、脳脊髄液中及び／又は血液中のHHV-6A、HHV-6B及びHHV-7の各ウイルスDNA量を健常人レベル以下、望ましくは検知水準以下に低下させることを目的とし、例えばホスカルネットの有効量として60～180mg/kg/日を、1日1回又は3回に分けて、髄腔内投与あるいは点滴静注により３日ないし10日間、望ましくは3ないし5日間継続して投与する。
　尚、ウイルスのDNA量の減少及びその維持状況、患者の認知機能の回復の状況を勘案し、並びにホスカルネットの忍容性の維持又は腎障害などの重篤な副作用の発生を防止もしくはコントロールする為、先制療法の期間及びホスカルネットの1回当たり又は1日当たりの投与量を適宜調整することができる。
［７］前記医薬組成物がホスカルネットを有効成分とする注射剤からなり、各ウイルスの再活性化の防止を目的とし、ホスカルネットの有効量として60～120mg/kg/日を、静注、筋注又は皮下注で、2ないし6ヵ月間継続して投与することによる、先制療法の後の維持療法のための医薬組成物である、前記［５］に記載の医薬組成物及びその使用が例示できる。
　ここで、本発明のホスカルネットかを有効成分とする注射剤をADの先制療法の後の維持療法として用いる場合、各ウイルスの再活性化の防止を目的として、例えば、ホスカルネットの有効量として60～120mg/kg/日を、静注、筋注又は皮下注で、望ましくは点滴静注で、２ないし6ヵ月間、望ましくは2～3ヵ月間、継続して投与する
　なお、ウイルスDNAの減少及びその維持状況、患者の認知機能の回復の状況を勘案し、並びにホスカルネットの忍容性の維持又は腎障害などの重篤な副作用の発生を防止もしくはコントロールする為、維持療法の期間並びにホスカルネットの1回当たり又は1日当たりの投与量を適宜調整することができる。
［８］前記医薬組成物がホスカルネットを有効成分とする経口剤からなり、注射剤による維持療法と共に／又はこれに代替して、ホスカルネットの１日当たり有効量として2000mgないし6000mgを、1日に3ないし5回に分けて、3ないし6ヵ月間、継続して投与することを特徴とする維持療法のための医薬組成物である、前記［５］記載の医薬組成物及びその使用が例示される。
　ここで、本発明のホスカルネットを有効成分とする経口剤を維持療法として用いる場合、注射剤による維持療法と共に／又はこれに代替して、ホスカルネットの１日当たり有効量として2000mgないし6000mgを、1日に3ないし5回に分けて、3ないし6ヵ月間、継続して投与する。また、ホスカルネットは粘膜刺激性を有することから、その経口剤の投与に際しては、テプレノン、スクラルファート、アズレンスルフォル酸ナトリウム、レパミピド、ポラプレジンク、イルソグラジンマイレン酸塩、ベキサネート塩酸塩、ソファルコン、セトラキサート塩酸塩、エカベトナトリウム水和物等の胃粘膜保護作用のある薬剤との併用あるいはそれらとの合剤が望ましい。
　なお、各ウイルスDNAの減少及びその維持状況、患者の認知機能の回復の状況を勘案し、並びにホスカルネットの忍容性の維持又は腎障害などの重篤な副作用の発生を防止もしくはコントロールする為、維持療法の期間並びに有効量のホスカルネットの1回当たり又は1日当たりの投与量を適宜調整することができる。
［９］ガンシクロビル、バラシクロビル、ペンシクロビル、ブリブジンからなる群から選択された少なくとも1つのヌクレオシド類縁体；シドフォビルもしくはそのプロドラッグ、誘導体又は他のヌクレオタイド類縁体の製剤；ヌクレオシド化合物の製剤；非ヌクレオシドDNAポリメラーゼ抑制化合物の製剤；アメナメビルもしくは他のヘリカーゼ・プライマーゼ阻害化合物の製剤；ウイルスDNAカプシドへのパッケージ阻害化合物；レテルモビルもしくは他のウイルスDNAターミナーゼ複合体の阻害剤；ニボルマブもしくは他のPD-1抗体、PD-1受容体抗体を含有する製剤の群から選択された少なくとも1つの製剤と併用して投与されることを特徴とする、前記［５］に記載の医薬組成物及びその使用。
　ここで、本発明のホスカルネットを有効成分とするADを治療（予防を含む）する為の医薬組成物は、化学的に又は作用メカニズムの異なる、脳炎などの中枢神経疾患を惹起するウイルスに対する抗ウイルス作用を有する化合物の製剤と併用して投与することができる。その際、同時に投与しても、別々に投与してもよい。
　併用できる化合物の製剤には、ガンシクロビル、バラシクロビル、ペンシクロビル、ブリブジなどのヌクレオシド類縁体の製剤、シドフォビルもしくはそのプロドラッグ、誘導体などのヌクレオタイド類縁体の製剤、ヌクレオシド化合物の製剤、非ヌクレオシドDNAポリメラーゼ抑制化合物の製剤、アメナメビルなどのヘリカーゼ・プライマーゼ阻害化合物の製剤、ウイルスDNAカプシドへのパッケージ阻害化合物、レテルモビルのようなウイルスDNAターミナーゼ複合体の阻害剤、ニボルマブのようなPD-1抗体、PD-1受容体抗体を含有する製剤があるが、これらに限定されない。
［１０］ドネペジル、ガランタミン、リバスチグミン、もしくはメマンチン製剤、神経保護剤、神経伝達物質からなる群から選択された少なくとも1つのAD治療剤又は神経伝達物質の移動の調整剤；アミロイドβもしくはAβオリゴマーを標的とする薬剤；タウタンパク質を標的とする薬剤；細胞への過剰なカルシウム流入抑制剤；ワクチン、イブプロフェン、ギンコエキス、もしくは他の抗炎症剤；インスリンもしくは他の抗糖尿病薬；抗IL-6抗体、抗IL-12抗体、もしくは他の炎症性サイトカイン、ケモカインの中和剤の群から選択された少なくとも1つの製剤と併用して投与されることを特徴とする、前記［５］に記載の医薬組成物及びその使用。
　ここで、本発明のホスカルネットを有効成分とするADを治療（予防を含む）する為の注射剤又は経口剤（調布剤、吸入剤を含む）は、ホスカルネットと作用メカニズムの異なる他のAD治療薬（「AD治療剤」）と併用して投与してもよい。
　併用するAD治療剤には、ドネペジル、ガランタミン、リバスチグミン、メマンチンの製剤の他、神経保護剤、神経伝達物質やその調整剤、アミロイドβ（オリゴマーを含む）を標的とする薬剤、タウタンパク質を標的とする薬剤、細胞内への過剰なカルシウム流入抑制剤、ワクチン、イブプロフェンやギンコエキスのような抗炎症剤、インスリンのような抗糖尿病薬、抗IL-6抗体、抗IL-12抗体などの炎症性サイトカイン、ケモカインの中和剤があるが、これらに限定されない。
［１１］ADに罹患するリスクのある人もしくはADの進展状況の診断、又はADの治療に用いる抗ウイルス剤の治療効果を確認する為の診断薬であって、脳脊髄液中、血漿中もしくは血液中もしくは唾液中のHHV-6Ａ及びHHV-6Ｂ並びにHHV-7のそれぞれに特異的なウイルスDNAをバイオマーカーとして含む診断薬及びその使用に用いることができる可能性がある。
　本発明により、ADが乳幼児期に「HHV-6」及び「HHV-7」に初感染し、一部の人は軽い炎症性の反応（HHV-6脳炎、知恵熱）を惹起するが、早期に治癒され大過なく時が経過する。
　初感染後、海馬や大脳辺縁系の細胞に潜伏感染した「HHV-6」が加齢に伴う免疫力の低下によって再活性化が更新して発症するHHV-6脳炎様の疾患であることが解明される。その為、脳脊髄液中もしくは血液中、望ましくは血漿中、更に望ましくは唾液中のHHV-6A及びHHV-6B並びにHHV-7のそれぞれに特異的なウイルスDNAをバイオマーカーとして用いることで、ADに罹患するリスクのある人、もしくはADの進展状況の診断及びADの治療に用いる抗ウイルス剤の治療効果を確認することができる。その際、アミロイドPET、タウPET、MRIなどADに関するバイオマーカーや診断方法を併用することで、より確実な診断が可能となる。 [6] The pharmaceutical composition comprises an injection containing foscarnet as an active ingredient, and the effective amount of foscarnet is 60 to 180 mg/kg/day divided into two or three times a day, and the medullary cavity The pharmaceutical composition according to the above [5] and its use, which are pharmaceutical compositions for preemptive therapy by continuous administration for 3 to 10 days by internal administration or intravenous drip infusion, can be exemplified.
Here, when the injection containing the foscarnet of the present invention as an active ingredient is used as a preemptive therapy for AD, each virus of HHV-6A, HHV-6B and HHV-7 in cerebrospinal fluid and/or blood is used. For the purpose of reducing the amount of DNA below the level of healthy subjects, preferably below the level of detection, an effective dose of foscarnet, for example, 60 to 180 mg/kg/day, is divided into 1 or 3 times a day, Administration by intracavitary injection or intravenous drip infusion for 3 to 10 days, preferably 3 to 5 days.
In addition, in consideration of the decrease in the amount of viral DNA and its maintenance status, the status of recovery of cognitive function in patients, and to prevent or control the occurrence of serious side effects such as maintenance of tolerability of foscarnet or renal damage. Therefore, the period of preemptive therapy and the dose of foscarnet per day or per day can be appropriately adjusted.
[7] The pharmaceutical composition comprises an injectable preparation containing foscarnet as an active ingredient, and an effective dose of foscarnet is 60 to 120 mg/kg/day for the purpose of preventing reactivation of each virus. Injection, intramuscular injection or subcutaneous injection, which is a pharmaceutical composition for maintenance therapy after pre-emptive therapy by continuous administration for 2 to 6 months, and the pharmaceutical composition according to the above [5] The use can be illustrated.
Here, in the case of using an injection containing the foscarnet of the present invention as an active ingredient as a maintenance therapy after AD preemptive therapy, for the purpose of preventing reactivation of each virus, for example, the effectiveness of foscarnet The dose is 60 to 120 mg/kg/day, administered intravenously, intramuscularly or subcutaneously, preferably intravenous drip for 2 to 6 months, preferably 2 to 3 months. Maintenance therapy in consideration of the decrease and maintenance status of DNA, the recovery status of patient's cognitive function, and to prevent or control the occurrence of serious side effects such as maintenance of tolerability of foscarnet or renal damage. And the dosage of foscarnet per dose or per day can be adjusted appropriately.
[8] The pharmaceutical composition comprises an oral agent containing foscarnet as an active ingredient, and together with/or in place of maintenance therapy by injection, an effective amount of foscarnet per day is 2000 mg to 6000 mg. A pharmaceutical composition for maintenance therapy, which is characterized in that it is continuously administered for 3 to 6 months in 3 to 5 divided doses, and the pharmaceutical composition and its use according to [5] above. It is illustrated.
Here, when an oral preparation containing the foscarnet of the present invention as an active ingredient is used as a maintenance therapy, in combination with or in place of the maintenance therapy by injection, an effective amount of foscarnet per day is 2000 mg to 6000 mg. , 3 to 5 times a day, continuously for 3 to 6 months. In addition, since foscarnet has mucosal stimulant properties, teprenone, sucralfate, sodium azulensulforate, repamipide, polaprezinc, irsogladine maleate, bexanate hydrochloride, sofarcone, when administering its oral preparation, A combination with a drug having a gastric mucosal protective action such as cetraxate hydrochloride, ecabet sodium hydrate or the like or a combination thereof is desirable.
In addition, in consideration of the decrease and maintenance status of each viral DNA and the status of recovery of cognitive function of patients, and to prevent or control the occurrence of serious side effects such as maintenance of tolerability of foscarnet or renal damage. Therefore, the duration of maintenance therapy and the dose of the effective amount of foscarnet per administration or per day can be appropriately adjusted.
[9] At least one nucleoside analog selected from the group consisting of ganciclovir, valacyclovir, penciclovir, brivudine; preparation of cidofovir or its prodrug, derivative or other nucleotide analog; preparation of nucleoside compound; non-nucleoside DNA polymerase Formulation of inhibitory compounds; Formulation of amenamevir or other helicase primase inhibitor compounds; Package inhibitor compounds to viral DNA capsids; Inhibitors of letermovir or other viral DNA terminase complexes; Nivolumab or other PD-1 antibodies, PD The pharmaceutical composition and its use according to the above [5], which is administered in combination with at least one preparation selected from the group of preparations containing -1 receptor antibody.
Here, the pharmaceutical composition for treating (including prevention) AD having foscarnet as an active ingredient of the present invention is chemically or different in action mechanism against viruses that cause central nervous system diseases such as encephalitis. It can be administered in combination with a formulation of a compound having an antiviral effect. At that time, they may be administered simultaneously or separately.
Preparations of compounds that can be used in combination include preparations of nucleoside analogs such as ganciclovir, valacyclovir, penciclovir and brivudi, preparations of nucleotide analogs such as cidofovir or its prodrugs, derivatives, preparations of nucleoside compounds, non-nucleoside DNA polymerase inhibitory compounds. , A helicase-primase inhibitor compound such as amenamevir, a package inhibitor compound for viral DNA capsid, an inhibitor of viral DNA terminase complex such as letermovir, PD-1 antibody such as nivolumab, PD-1 receptor There are, but are not limited to, formulations containing body antibodies.
[10] At least one AD therapeutic agent or neurotransmitter migration regulator selected from the group consisting of donepezil, galantamine, rivastigmine, or memantine preparations, neuroprotective agents, and neurotransmitters; targeting amyloid β or Aβ oligomers Drug; drug targeting tau protein; inhibitor of excessive calcium influx into cells; vaccine, ibuprofen, ginkgo extract, or other anti-inflammatory drug; insulin or other antidiabetic drug; anti-IL-6 antibody, The pharmaceutical composition according to the above [5], which is administered in combination with at least one preparation selected from the group consisting of anti-IL-12 antibody, other inflammatory cytokine, and a chemokine neutralizing agent. Things and their use.
Here, an injection or an oral preparation (including a fabric preparation and an inhalant) for treating (including preventing) AD containing foscarnet as an active ingredient of the present invention has a different action mechanism from foscarnet. It may be administered in combination with the AD therapeutic agent ("AD therapeutic agent").
In addition to preparations of donepezil, galantamine, rivastigmine, memantine, neuroprotective agents, neurotransmitters and their regulators, agents that target amyloid β (including oligomers), and tau protein are targeted Drugs, inhibitors of excessive calcium influx into cells, vaccines, anti-inflammatory agents such as ibuprofen and ginkgo extract, anti-diabetic agents such as insulin, inflammatory properties such as anti-IL-6 antibody and anti-IL-12 antibody There are, but are not limited to, cytokines, chemokines neutralizing agents.
[11] A diagnostic agent for diagnosing a person at risk of suffering from AD or the progress of AD, or for confirming the therapeutic effect of an antiviral agent used for the treatment of AD, which is in cerebrospinal fluid, plasma or There is a possibility that it can be used for a diagnostic agent containing viral DNAs specific for HHV-6A and HHV-6B in blood or saliva and HHV-7 as biomarkers and their use.
ADVANTAGE OF THE INVENTION According to the present invention, AD is initially infected with "HHV-6" and "HHV-7" in infancy, and some people elicit a mild inflammatory reaction (HHV-6 encephalitis, wisdom fever), but at an early stage. Healed and time passed without any major mistake.
It is a HHV-6 encephalitis-like disease in which "HHV-6" latently infected to hippocampus and limbic cells after the initial infection develops due to reactivation renewed due to a decrease in immunity with age. To be elucidated. Therefore, in the cerebrospinal fluid or blood, preferably in plasma, more preferably by using the viral DNA specific for HHV-6A and HHV-6B and HHV-7 in saliva as biomarkers, AD It is possible to confirm the therapeutic effect of an antiviral agent used for diagnosing the progress of AD and treating AD, for persons at risk of suffering from AD. At that time, by using a biomarker and a diagnostic method for AD such as amyloid PET, tau PET, and MRI together, a more reliable diagnosis becomes possible.
　現時点で、ADに罹患しあるいは発症した場合、その進行を阻止しあるいは回復させる根本的な治療薬は開発されていない。
　ADで最初に障害を受けるのは、神経細胞の再生、分裂が行われ、早期記憶や認知機能を掌る海馬とその周辺の神経組織である。かかる障害の主因である海馬や周辺組織でのウイルス増殖を抑制により、再生能のある神経細胞による障害の修復が可能となる。その結果、ADの進行を阻止するだけでなく回復あるいは根治への可能性が期待できる。
　本発明は、ADの症状（特に記憶・認知障害）を緩和するだけの対症療法薬ではなく、AD発症の根本的原因の一つである「HHV-6」及び「HHV-7」の再活性化を阻止することによるADの根本的治療あるいは予防が期待できる。
　以上により、ADの治療に対する医療費の増大を抑え、また、ADによる休職や介護等に伴う社会的負担、損失を大幅に低減できる。 At this time, no underlying therapeutic agent has been developed to prevent or reverse the progression of AD if it suffers or develops.
The first lesions in AD are the hippocampus and its surrounding nerve tissue, which are responsible for the regeneration and division of nerve cells and are responsible for early memory and cognitive functions. By suppressing the proliferation of the virus in the hippocampus and the surrounding tissues, which is the main cause of such disorders, it becomes possible to repair the disorders by nerve cells having a regenerative ability. As a result, not only the progress of AD can be prevented, but also the possibility of recovery or cure can be expected.
The present invention is not a symptomatic drug that only relieves the symptoms of AD (particularly memory/cognitive impairment), but reactivation of "HHV-6" and "HHV-7" that is one of the underlying causes of AD development. The treatment or prevention of AD can be expected to be prevented by preventing the change.
From the above, it is possible to suppress an increase in medical expenses for the treatment of AD, and it is possible to significantly reduce the social burden and loss associated with leave and nursing care due to AD.
Aβ仮説の概略を示した図（非特許文献１より引用）。現在、AD発症の機序として主流となっている仮説The figure showing the outline of the Aβ hypothesis (cited from Non-Patent Document 1). Hypothesis currently the mainstream mechanism of AD development AD発症に関するHHV-6脳炎カスケード仮説」の概略（Aβが関与する機序）とそれに対するマルチ抗ウイルス剤の作用点を示した図（非特許文献１より改変）Figure showing the outline of "HHV-6 encephalitis cascade hypothesis regarding AD onset" (mechanism in which Aβ is involved) and the action points of multi-antiviral agents against it (modified from Non-Patent Document 1) ホスカルネットと他の抗ウイルス剤との比較を示した図で、非特許文献１６のFigure 1 から引用。HHV-6A、HHV-6B及びHHV-7に対するホスカルネット（PFA）、アシクロビル（ACV）、パンシクロビル（PCV）、ガンシクロビル（GCV）などとの抗ウイルス効果（EC50）を比較した試験で、ホスカルネットはこれら3ウイルスに対し、30μμg/mLの範囲内で効果を示し、また、HHV-6A及びHHV-7に対し、HHV-6Bの約1／2の用量で効果を示しており、ACV、PCV、GCVに比べてバランスの取れた薬効プロファイルを持つ。A diagram showing a comparison between foscarnet and other antiviral agents, taken from Figure 1 of Non-Patent Document 16. In a study comparing antiviral effects (EC 50 ) with foscarnet (PFA), acyclovir (ACV), pancyclovir (PCV), ganciclovir (GCV), etc. against HHV-6A, HHV-6B and HHV-7 , Foscarnet was effective against these three viruses within the range of 30 μg/mL, and was effective against HHV-6A and HHV-7 at about 1/2 the dose of HHV-6B. Has a balanced efficacy profile compared to ACV, PCV and GCV. 非特許文献１４のFig 2からの引用である。ホスカルネット4000mgを含有する水溶液を1日3回、3日間、AID患者6名に経口投与後の血漿中及び脳脊髄液中の濃度の変化を示す図である。投与直後には血漿中の濃度はピークの達し、6時間後にはピーク時の約1／3に、12時間後には約1／10に低下する。しかし、脳脊髄液中では、投与約1時間後にピーク（血漿中濃度の約25%）に達し、以降、緩徐に低下し、6時間後には血漿中濃度の80%以上を維持し、12時間後には血漿中の濃度を上回る。尚、12時間でも、HHV-6（A及びB）、HHV-7のIC50を確保できる濃度（50μM/L）を保っている。（「Raffi F, Antimicrob Agents Chemother 1993, 37(9)1777-80」から引用）This is a citation from Fig. 2 of Non-Patent Document 14. FIG. 2 is a graph showing changes in plasma and cerebrospinal fluid concentrations after oral administration of an aqueous solution containing 4000 mg of foscarnet to 6 AID patients 3 times a day for 3 days. The concentration in plasma reaches a peak immediately after the administration, and decreases to about 1/3 of the peak after 6 hours and to about 1/10 after 12 hours. However, in cerebrospinal fluid, it reached a peak (about 25% of plasma concentration) about 1 hour after administration, and then decreased slowly, and after 6 hours, maintained at 80% or more of plasma concentration for 12 hours. Later it exceeds the concentration in plasma. Even for 12 hours, HHV-6 (A and B) and HHV-7 are kept at a concentration (50 μM/L) that can ensure an IC 50 . (Quoted from "Raffi F, Antimicrob Agents Chemother 1993, 37(9)1777-80") ヒトにホスカルネット製剤「ホスカビル（登録商標）」を投与したときの血中ホスカルネット濃度とカルシウムイオン濃度との関係を示す。ヒトにホスカビル（登録商標）（ホスカルネットとして90mg/kg/12時間）を反復点滴静注したときの血中でのホスカルネット濃度とカルシウム・イオン濃度に負の相関関係が認められた（r=0.96: p<0.001）が、カルシウムイオン濃度の低下は用量に依存した一過性の変化で、点滴終了後ホスカルネットの濃度の低下とともに徐々に正常値に戻っている。（「点滴静注用ホスカビル（登録商標）注24mg/mL インタビューフォーム2019年3月版」から引用）1 shows the relationship between blood foscarnet concentration and calcium ion concentration when a foscarnet preparation “foscavir (registered trademark)” was administered to humans. A negative correlation was observed between the foscarnet concentration in blood and the calcium ion concentration after repeated intravenous infusion of foscavir (registered trademark) (90 mg/kg/12 hours as foscarnet) in human ( r=0.96: p<0.001), however, the decrease in calcium ion concentration was a dose-dependent transient change, and gradually returned to normal with the decrease in foscarnet concentration after the end of infusion. (Quoted from “Foscavir (registered trademark) Note 24 mg/mL Interview Form March 2019 Edition for Intravenous Infusion”) ホスカビル（登録商標）の濃度勾配によるβセクレターゼ阻害の確認（N=1）：ホスカビル（登録商標）のBASE-1の酵素阻害作用を調べた試験（実施例４）の結果を示した図。ホスカビル（登録商標）のBASE-1酵素阻害活性を標準的な試験用抗体とホスカビル（登録商標）の溶液とを比較したところ、ホスカビル（登録商標）の溶液は濃度変化に応じた阻害効果を示したが、その値は低かった。Confirmation of β-secretase inhibition by a concentration gradient of foscavir (registered trademark) (N=1): a diagram showing the results of a test (Example 4) in which the enzyme inhibitory effect of foscavir (registered trademark) on BASE-1 was examined. The BASE-1 enzyme inhibitory activity of foscavir (registered trademark) was compared between the standard test antibody and the foscavir (registered trademark) solution, and the foscavir (registered trademark) solution showed an inhibitory effect according to the concentration change. However, the value was low. ホスカビル（登録商標）の濃度勾配によるβセクレターゼ阻害の確認（N=2）Confirmation of β-secretase inhibition by a concentration gradient of foscavir (N=2) ヒト神経芽細胞腫株（「SH-SY5Y株」）を用い、HHV-6/7の存在下及び非存在下で、Aβ42が細胞の増殖に及ぼす影響を調べた試験結果（１）。培養開始から48時間後に、いずれの試験群でも細胞数が増えていることから、細胞外Aβ42の被験細胞に対する毒性は観察されていない。A test result (1) using a human neuroblastoma cell line (“SH-SY5Y strain”) to examine the effect of Aβ 42 on cell proliferation in the presence and absence of HHV-6/7. No toxicity of extracellular Aβ 42 to the test cells was observed since the number of cells increased in any of the test groups 48 hours after the start of culture. ヒト神経芽細胞腫株（「SH-SY5Y株」）を用い、HHV-6/7の存在下及び非存在下で、Aβ42が細胞の増殖に及ぼす影響を調べた試験結果（２）。A test result (2) using a human neuroblastoma cell line (“SH-SY5Y cell line”) to investigate the effect of Aβ 42 on cell proliferation in the presence and absence of HHV-6/7. SH-SY5Y株を用い、HHV-6/7の存在下においてAβ42が細胞内に取り込まれるか否かを確認した試験の結果を示す図。10nM以下のAβ42添加条件では添加濃度に依存した細胞内Aβ42量が示された。また、HHV-7とHHV-6Aのウイルス溶液の添加条件（D群）、HHV-7とHHV-6Bのウイルス溶液の添加条件（E群）においても細胞内Aβ42量は減少傾向にあった。これらの事実は、細胞外ウイルスがAβ42の細胞内取り込みを抑制する可能性を示唆した。　また、ホスカルネット添加群（A及びF～H）では、未添加群（A及びC～E群）と比較して細胞内Aβ42量に明確な差はなかった。The figure which shows the result of the test which confirmed whether A(beta) 42 is taken up into a cell in presence of HHV-6/7 using SH-SY5Y strain|stump|stock. Under the condition of Aβ 42 addition of 10 nM or less, intracellular Aβ 42 amount depending on the addition concentration was shown. In addition, the intracellular Aβ 42 content also tended to decrease under the addition conditions of HHV-7 and HHV-6A virus solution (Group D) and HHV-7 and HHV-6B virus solution (Group E). .. These facts suggested that extracellular virus may suppress intracellular uptake of Aβ 42 . In addition, in the foscarnet-added group (A and F to H), there was no clear difference in the intracellular Aβ 42 amount as compared with the non-added group (A and C to E groups). 試験ウイルスに感染したSH-SY5Y株内のウイルスDNA量を調べた試験の結果を示す図。（図９Ａ及びＢ）では試験ウイルスとしてはHHV-6を用いている。The figure which shows the result of the test which investigated the viral DNA amount in SH-SY5Y strain infected with the test virus. In FIGS. 9A and 9B, HHV-6 is used as the test virus. 試験ウイルスに感染したSH-SY5Y株内のウイルスDNA量を調べた試験の結果を示す図。（図９Ｃ及びＤ）では試験ウイルスとしてはHHV-7を用いている。The figure which shows the result of the test which investigated the viral DNA amount in SH-SY5Y strain infected with the test virus. In FIGS. 9C and D, HHV-7 is used as the test virus. 実施例５の中でヒト由来リンパ球株（HSB-2株）について、HHV-6Aが及ぼす影響を調べた試験の結果を示した図。ウイルス溶液の未添加群（左図）とHHV-6Aウイルス溶液を添加した群（右図）に分けて培養したところ、（左図）では細胞に何らの変化も生じないが、ウイルス溶液を添加した群（右図）では、アポトーシスする細胞に特有な形状（ゴツゴツした細胞）、ネクローシスする細胞に特有な形状（膨張等）、未変化の細胞、死亡細胞の断片が認められている。図１０～１２の細胞写真の全てにおいて、非感染の細胞と比較し、ウイルス添加細胞ではゴツゴツした細胞、径の大きな細胞、死細胞が散見されるが、これらはウイルス感染により変化した細胞と考えられる。The figure which showed the result of the test which investigated the influence which HHV-6A has about the human-derived lymphocyte strain (HSB-2 strain) in Example 5. When the cells were cultured separately in the group that did not add the virus solution (left figure) and the group that added the HHV-6A virus solution (right figure), no change was observed in the cells in the (left figure), but the virus solution was added. In the group (right), specific shapes of apoptotic cells (ragged cells), specific shapes of necrotic cells (swelling, etc.), unchanged cells, and dead cell fragments were observed. In all the cell photographs of FIGS. 10 to 12, in comparison with non-infected cells, rugged cells, large-diameter cells, and dead cells are found scatteredly in the virus-added cells, which are considered to be cells changed by virus infection. To be 実施例５の中でヒト由来Ｔ細胞株（Sup-T1株）について、HHV-6Bが及ぼす影響を調べた試験の結果を示す図。ウイルス溶液の未添加の群（左図）、HHV-6Bウイルス溶液を添加した群（右図）に分けて培養したところ、（左図）では試験細胞に何らの変化も生じていない。しかし、各ウイルス溶液を添加した（右図）では、アポトーシスする細胞に特有な形状（ゴツゴツした細胞）、ネクローシスする細胞に特有な形状（膨張等）、未変化の細胞、死亡細胞の断片が認められた。FIG. 5 is a diagram showing the results of a test for examining the effect of HHV-6B on the human-derived T cell line (Sup-T1 strain) in Example 5. When the cells were cultured separately in the group to which the virus solution was not added (left figure) and the group to which the HHV-6B virus solution was added (right figure), there was no change in the test cells in the (left figure). However, when each virus solution was added (right panel), a shape unique to apoptotic cells (cracked cells), a shape unique to necrotic cells (swelling, etc.), unchanged cells, and dead cell fragments were observed. Was given. 実施例５の中でヒト由来Ｔ細胞株（Sup-T1株）について、HHV-7が及ぼす影響を調べた試験の結果を示す図。図１０，１１と同様に、アポトーシス又はネクローシスする細胞に特有の形状の細胞が複数観察された。FIG. 5 is a diagram showing the results of a test for examining the effect of HHV-7 on the human-derived T cell line (Sup-T1 strain) in Example 5. Similar to FIGS. 10 and 11, a plurality of cells having a shape unique to the cells undergoing apoptosis or necrosis were observed. 実施例５の中でHSB-2株にHHV-6Aが感染するか否かを抗HHV-6A gp82抗体を用いる免疫染色法で調べた試験の結果を示した図。HHV-6Aウイルス溶液の未添加群（左図）と添加した群（右図）についウイルス感染の有無を確認したところ、（左図）では抗体による陽性反応（DABによる茶褐色の染色）は見られなかったが、（右図）では多くの細胞が陽性反応を示した。FIG. 5 is a diagram showing the results of a test conducted in Example 5 to examine whether HSB-2 strain is infected with HHV-6A by an immunostaining method using an anti-HHV-6A gp82 antibody. When the presence or absence of virus infection was confirmed in the group without addition of HHV-6A virus solution (left figure) and the group with addition (right figure), positive reaction with antibody (dark brown staining with DAB) was observed in (left figure). However, many cells showed a positive reaction in (right panel). 実施例５の中でヒト由来Ｔ細胞株（Sup-T1株）にHHV-6Bが感染するか否かを抗HHV-6B gp98抗体による免疫染色法で調べた試験の結果を示した図。HHV-6Bウイルス溶液の未添加群（左図）と添加した群（右図）についウイルス感染の有無を確認したところ、（左図）では抗体による陽性反応（DABによる茶褐色の染色）は見られなかったが、（右図）では多くの細胞が陽性反応を示した。なお、図１３Ｂ及びＣの免疫染色法による感染試験結果から、HHV-7よりもHHV-6Bの方がSup-T1細胞に対する感染力が強いことが示された。The figure which showed the result of the test investigated by the immunostaining method with an anti-HHV-6B gp98 antibody whether or not HHV-6B infects a human-derived T cell line (Sup-T1 strain) in Example 5. When the presence or absence of virus infection was confirmed in the group without addition of HHV-6B virus solution (left figure) and the group with addition (right figure), positive reaction with antibody (dark brown staining with DAB) was observed in (left figure). However, many cells showed a positive reaction in (right panel). The infection test results by the immunostaining method shown in FIGS. 13B and 13C showed that HHV-6B had stronger infectivity to Sup-T1 cells than HHV-7. 実施例５の中でヒト由来Ｔ細胞株（Sup-T1株）にHHV-7が感染するか否かをHHV-7KR4抗体による免疫染色法で調べた試験の結果を示した図。HHV-7ウイルス溶液の未添加添群（左図）とHHV-6Bウイルス添加群（右図）について、免疫染色法で感染の有無を確認したところ、（左図）では抗体による陽性反応（DABによる茶褐色の染色）は見られなかったが、（右図）では多くの細胞が陽性反応を示した。FIG. 7 shows the results of a test conducted in Example 5 to examine whether or not HHV-7 is infected with a human-derived T cell line (Sup-T1 strain) by an immunostaining method using an HHV-7KR4 antibody. The presence or absence of infection was confirmed by immunostaining in the HHV-7 virus solution-free group (left figure) and HHV-6B virus-containing group (right figure). However, many cells showed a positive reaction in (right panel). 脳内で潜伏感染しているHHV-6/7の再活性化に伴ってADが発症するという「HHV-6脳炎カスケード仮説」によるAD発症プロセスの概要を示した図。The figure which showed the outline of the AD onset process by "HHV-6 encephalitis cascade hypothesis" that AD develops with reactivation of latently infected HHV-6/7 in the brain.
（２－１）HHV-6（A及びB）及び／又はHHV-7に対する抗ウイルス剤
　本発明のアルツハイマー病（AD）に治療及び予防の為の医薬組成物としては、HHV-6（A及びB）やHHV-7に対する抗ウイルス効果を有する化合物やその医薬組成物（製剤）が有効である。
　そのような化合物、製剤としては、ホスカルネット（特表平１０－５０９７０４号、特表平１１－５０９５１５号、特開２００７－２８４４５２号）、ファムシクロビル（特開平０４－２７５２２９号）、アシクロビル（米国特許第４９５７９２４4号）、バラシクロビル（特許第３３５００５５号）、ガンシクロビル（特開平０８－５３４５２号）、シドフォビルを含むフォスフォネート誘導体（特許第５９６３７８７号）、抗CD40抗体（特表２００２－５４３１５０号）、ピリドキサノサリン（特表２００５－５１６９５７号）、ピラゾロピリジン誘導体（特表２００４－５２９１１９号公報）、SITH-1阻害剤（再表２００９－０４１５０１号）、ナノ・エマルジョン組成物（特開２０１１－５１８１８４号）、グレリン誘導体（特開２０１８－１３８５８６号）などがある。これらの化合物のうち、ホスカルネット及びガンシクロビルは、HHV-6脳炎に対しても適用され有効性が確認されており、ADの治療及び予防用医薬組成物の有効成分として好ましい。特に好ましいのは、マルチ抗ウイルス剤として知られるホスカルネットである。また、これら化合物を２つ以上併用して、合剤として又は組み合わせ剤などとして用いてもよい。 (2-1) Antiviral agent against HHV-6 (A and B) and/or HHV-7 As a pharmaceutical composition for treating and preventing Alzheimer's disease (AD) of the present invention, HHV-6 (A and B Compounds having an antiviral effect against B) and HHV-7 and pharmaceutical compositions (formulations) thereof are effective.
Examples of such compounds and preparations include foscarnet (Japanese Patent Publication No. 10-509704, Japanese Patent Publication No. 11-509515, JP 2007-284452), famciclovir (JP 04-275229), acyclovir. (US Pat. No. 4,957,244), valacyclovir (Patent No. 3350055), ganciclovir (Japanese Patent Laid-Open No. 08-53452), phosphonate derivative containing cidofovir (Patent No. 5963787), anti-CD40 antibody (Japanese Patent Publication No. 2002-543150). ), pyridoxanosaline (Table 2005-516957), pyrazolopyridine derivative (Table 2004-527119), SITH-1 inhibitor (Table 2009-041501), nanoemulsion composition (special No. 2011-518184), ghrelin derivative (JP-A-2018-138586) and the like. Of these compounds, foscarnet and ganciclovir have been confirmed to be effective when applied to HHV-6 encephalitis, and are preferable as active ingredients of a pharmaceutical composition for treating and preventing AD. Particularly preferred is foscarnet, which is known as a multi-antiviral agent. Further, two or more of these compounds may be used in combination and used as a mixture or a combination.
（２－２）ホスカルネットについて
　本発明で「ホスカルネット」というとき、下記（式１）で示されるホスホノギ酸もしくはその塩、又はこれらの溶媒和物を指す。ここで、塩としては、医薬として許容され得る塩であり、リチウム塩、ナトリウム塩、カリウム塩、マギネシウム塩、カルシウム塩等の金属塩、アンモニウム塩、メチルアンモニウム塩、ジメチルアンモニウム塩、トリメチルアンモニウム塩、ジシクロヘキシルアンモニウム塩等のアンモニウム塩などの他、塩酸塩、臭酸塩、硫酸塩、硝酸塩、リン酸、メタリン酸塩等等の鉱産塩、メタンスルホン酸塩、ベンゼンスルホン酸塩、パラトルエンスルホン酸塩、酢酸塩、プロピオン酸塩、酒石酸塩、フマル酸塩、マイレン酸塩、リンゴ酸塩、シュウ酸塩、コハク酸塩、クエン酸塩、安息香酸塩、マンデル酸塩、ケイヒ酸塩、乳酸、ベシル酸、吉草酸、ステアリン酸、オイレン酸、グルコン酸、ラウリル酸、サルチル酸、ラオクル酸、タンニン酸、ブチルスルホン酸塩等の有機酸塩などを形成することができる。溶媒和酸を形成し得る溶媒としては、メタノール、エタノール、イソプロパノール、アセトン、酢酸エチル、塩化メチレン、ジイソプロピルエーテル等が挙げられる。 (2-2) Foscarnet When the term "foscarnet" is used in the present invention, it means phosphonoformic acid represented by the following (formula 1) or a salt thereof, or a solvate thereof. Here, the salt is a pharmaceutically acceptable salt, such as lithium salt, sodium salt, potassium salt, magnesium salt, metal salt such as calcium salt, ammonium salt, methylammonium salt, dimethylammonium salt, trimethylammonium salt, In addition to ammonium salts such as dicyclohexyl ammonium salt, mineral salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphoric acid, metaphosphate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc. , Acetate, propionate, tartrate, fumarate, maleate, malate, oxalate, succinate, citrate, benzoate, mandelate, cinnamate, lactic acid, besil It is possible to form acids, valeric acid, stearic acid, oleic acid, gluconic acid, lauric acid, salicylic acid, laocric acid, tannic acid, organic acid salts such as butyl sulfonate, and the like. Examples of the solvent capable of forming a solvated acid include methanol, ethanol, isopropanol, acetone, ethyl acetate, methylene chloride, diisopropyl ether and the like.
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
　そのうちの最も好ましい化合物は、下記（式２）で表されるホスカルネットナトリウム水和物であり、(JNN)/INN表記でFoscarnet Sodiumと表記される。以下、典型的な「ホスカルネット」として、主として（式２）の化合物について述べるが、これに限定されるものではない。 The most preferred compound among them is foscarnet sodium hydrate represented by the following (formula 2), and is represented as Foscarnet Sodium in (JNN)/INN notation. Hereinafter, the compound of the formula (2) will be mainly described as a typical “foscarnet”, but the invention is not limited thereto.
Figure JPOXMLDOC01-appb-C000007
 （式２）
Figure JPOXMLDOC01-appb-C000007
 (Formula 2)
ホスカルネットは、HHV-6やHHV-7に対する強い抗ウイルス作用を持ち、造血幹細胞や臓器移植後に発症するHHV-6脳炎に対する優れた臨床効果を有する化合物で、以下の特徴を有し、アルツハイマー病の治療（予防を含む）に向けた抗ウイルス剤として最も適している。 Foscarnet is a compound that has a strong antiviral action against HHV-6 and HHV-7 and has an excellent clinical effect on HHV-6 encephalitis that develops after hematopoietic stem cells and organ transplants, and has the following characteristics, and Alzheimer's Most suitable as an antiviral agent for the treatment (including prevention) of diseases.
（ａ）抗ウイルス作用
　HHV-6A及びHHV-7に対する約2倍の有効量でHHV-6Bに対する効果を有し、ウイルスごとに投与量を大きく変える必要がなく、バランスのとれた強い抗ウイルス作用を持つ。また、HSV-1やCMVに対しても、HHV-6A及びHHV-6B並びにHHV-7に対する有効量によって強い抗ウイルス作用を持つ（図３）。 (A) Antiviral action It has an effect on HHV-6B at an effective dose about twice that of HHV-6A and HHV-7, and it has a well-balanced and strong antiviral action without the need to significantly change the dose for each virus. have. It also has a strong antiviral effect against HSV-1 and CMV, depending on the effective amounts of HHV-6A, HHV-6B and HHV-7 (Fig. 3).
（ｂ）βセクレターゼの阻害作用
　アミロイドβ（Aβ）はアミロイド前駆体タンパク質（APP）がβセクレターゼとγセクレターゼという2種のアミノ酸分解酵素によって分解、生成される。
　本発明において、ホスカルネットはβセクレターゼ及び／又はγセクレターゼの酵素活性を阻害する作用が低いながら認められ（表４）、Aβの産生を抑制する作用が期待できる。 (B) Inhibitory action of β-secretase Amyloid β (Aβ) is produced by degrading amyloid precursor protein (APP) by two amino acid degrading enzymes, β-secretase and γ-secretase.
In the present invention, foscarnet has been found to have a low effect of inhibiting the enzyme activity of β-secretase and/or γ-secretase (Table 4), and an action of suppressing Aβ production can be expected.
（ｃ）アミロイドβ及びタウタンパク質の蓄積の阻害
　上記（ｂ）の作用に加え、抗ウイルス作用でウイルスの増殖を抑制すれば、Aβの過剰な産生や分泌、そのオリゴマー化を阻止でき、それだけでなく、蓄積したAβオリゴマーの活性化によって起きるタウタンパク質の過剰リン酸化の抑制も可能である。
　上記を確認する為、SH-SY5Y株（被験細胞）の培地にAβ42、HHV-6AとHHV-7の混合液、HHV-6BとHHV-7の混合液及びHHV-7単独溶液並びにホスカビル（登録商標）（800μM）を添加し、被験細胞内へのAβ42の取り込みを検証する為にその細胞内濃度を、ウイルスの取り込み（感染）を検証する為にウイルスDNA数を測定した。
　その結果、比較対照であるウイルス溶液不添加の細胞群と比べて、ウイルス溶液を添加した群では細胞内Aβ42量は減少傾向を示し、この減少傾向はウイルス溶液添加群にホスカビル（登録商標）を添加しても差はなかった（図７，８）。また、ホスカルビル添加群では細胞内HHV-6DNA数は添加Aβの増加に伴って減少していた。このことは、試験条件下において細胞外のAβ42は被験細胞内に取り込まれていること、Aβ42は試験した各ウイルスの細胞内取り込を抑制すること、ホスカルビルの添加はかかる抑制を阻害しないことを示した。
　以上の結果は、細胞外に出現したウイルスなどの病原体を捕捉するというAβの自然免疫作用（W Eimer et al., Neuron 99 (1): 56-63, 2018、RD Moir et al., Alzheimer’s & Dementias 14 (2018): 1602-1614, 201）はHHV-6/7感染にも適用されることを裏付ける証左といえた。また、ホスカビル（登録商標）の添加による細胞内ウイルスDNA数の減少効果は示されたが、それがホスカビル（登録商標）の抗ウイルス作用によるのか、あるいは「ウイルスAβ複合体」の細胞内への取り込みの抑制によるのかは、本試験条件では確認できなかった。しかし、細胞外Aβは濃度依存的（10nM以下の添加条件）に細胞内に取り込まれるという試験結果から、「ウイルスAβ複合体」の細胞内取り込みを抑制している可能性はある。 (C) Inhibition of accumulation of amyloid β and tau protein In addition to the action of (b) above, if the virus growth is suppressed by an antiviral action, excessive production and secretion of Aβ and its oligomerization can be prevented. Nonetheless, it is possible to suppress hyperphosphorylation of tau protein caused by activation of accumulated Aβ oligomers.
In order to confirm the above, in the medium of the SH-SY5Y strain (test cell), Aβ 42, a mixed solution of HHV-6A and HHV-7, a mixed solution of HHV-6B and HHV-7 and a single solution of HHV-7 and foscarvir ( (Registered trademark) (800 μM) was added, and its intracellular concentration was measured in order to verify the uptake of Aβ 42 into the test cells, and the number of viral DNA was measured in order to verify the virus uptake (infection).
As a result, the intracellular Aβ 42 amount showed a tendency to decrease in the group to which the virus solution was added, as compared with the cell group to which the virus solution was not added, which was a comparative control. There was no difference even after adding (Figs. 7 and 8). In the foscarvir-added group, the number of intracellular HHV-6 DNA decreased with the increase of added Aβ. This means that extracellular Aβ 42 is taken up into the test cells under the test conditions, Aβ 42 suppresses intracellular uptake of each virus tested, and addition of foscarvir does not inhibit such suppression. I showed that.
The above results show that Aβ's innate immunity that captures extracellular pathogens such as viruses (W Eimer et al., Neuron 99 (1): 56-63, 2018, RD Moir et al., Alzheimer's & Dementias 14 (2018): 1602-1614, 201) is proof that it is also applicable to HHV-6/7 infection. In addition, although the effect of reducing the intracellular viral DNA number by the addition of foscavir (registered trademark) was shown, whether it was due to the antiviral effect of foscavir (registered trademark), or the "virus Aβ complex" It could not be confirmed under the present test conditions whether it was due to inhibition of uptake. However, from the test results that extracellular Aβ is taken up intracellularly in a concentration-dependent manner (addition condition of 10 nM or less), it is possible that the intracellular uptake of “viral Aβ complex” is suppressed.
（ｄ）神経細胞死の抑制　　
　これまでHHV-6/7に感染した細胞はアポトーシスするとされていた（R Ichimi et al., Journal of Medical Virology, 58 (1): 63-68, 1999、及び非特許文献１０、参考文献３０，３１）ことから、上記試験に関連して各ウイルスによる感染細胞死の有無を検証した。（実施例５）
　HHV-6Aに関しては、未感染のHSB-2株（ヒトTリンパ球芽細胞株）、HHV-6B及びHHV-7に関して未感染のSup-T1株（ヒトTリンパ球由来T細胞株）に実施例１で得られた各ウイルス溶液を添加し、ウイルス感染の有無、感染細胞の形態の変化を観察した（実施例５）。
　その結果、HHV-6Aの未感染細胞群（図１０－１）では、各細胞の形や大きさは均一で、死亡した細胞は殆ど観察されていない。しかし、感染細胞群（図１０－２）では、肥大した細胞及び死細胞が散見された。
　また、HHV-6B及びHHV-7に関しては、未感染のSup-T1群（図１１－１）では、死細胞は見られず、細胞の形、大きさも均一である。しかし、HHV-6B感染細胞群（図１１－２）及びHHV-７感染細胞群（図１２）で僅かな死細胞の他に、細胞表面がゴツゴツした細胞、肥大した細胞が散見された。
　上記試験によって、HHV-6A、HHV-6B、HHV-7に感染した細胞には、細胞表面に凹凸が生じるもの、肥大化するという形態変化が起きることが明らかになった。この内、細胞表面に凹凸が生じた細胞は、その径が非感染細胞と変わらないこと、アポトーシスの場合は細胞膜の形質変化を起点とすることから、アポトーシス細胞を示している。
他方、細胞の肥大化はネクローシスの典型的な形態（参考文献２４）であることから、肥大化した細胞は、ネクローシスする細胞と考えられる。また、免疫染色で陽性反応を示す細胞は多いが、アポトーシス及びネクローシスとなる細胞数が少ないことから、感染細胞の多くは正常な状態（形態）を示すことが明らかになった。
　上記の試験により、HHV-6A、HHV-6B、HHV-7に感染した細胞はアポトーシスだけでなく、ネクローシスをも惹起することが初めて明らかにされた。
　以上の知見は、ADにおいて脳内、特に神経細胞の新生が行われている海馬において神経細胞やグリア細胞に潜伏感染しているHHV-6AやHHV-6Bが宿主細胞のアポトーシスを含む細胞死に際して再活性化した場合、ネクローシスをも惹起し、当該細胞からDAMPs（Damage/Danger Associated Molecular Patterns）として様々な炎症起因物質（例えばHMGB-1，TDP-43、GSK-3β、グルタミン酸、Ca＋＋等）が放出され、炎症が増強されることを示唆している。 (D) Suppression of nerve cell death
Until now, cells infected with HHV-6/7 were said to be apoptotic (R Ichimi et al., Journal of Medical Virology, 58 (1): 63-68, 1999, and Non-Patent Document 10, Reference Document 30, Reference Document 30, Therefore, the presence or absence of infected cell death by each virus was verified in relation to the above test. (Example 5)
For HHV-6A, performed on uninfected HSB-2 strain (human T lymphocyte blast cell line) and HHV-6B and HHV-7 uninfected Sup-T1 strain (human T lymphocyte-derived T cell line) Each virus solution obtained in Example 1 was added, and the presence or absence of virus infection and changes in the morphology of infected cells were observed (Example 5).
As a result, in the HHV-6A uninfected cell group (FIG. 10-1), the shape and size of each cell were uniform, and almost no dead cells were observed. However, in the infected cell group (FIG. 10-2), enlarged cells and dead cells were scattered.
Regarding HHV-6B and HHV-7, dead cells were not found in the uninfected Sup-T1 group (Fig. 11-1), and the cell shape and size were uniform. However, in the HHV-6B-infected cell group (FIG. 11-2) and the HHV-7-infected cell group (FIG. 12), in addition to a few dead cells, rugged cells and swelled cells were scattered.
The above test revealed that cells infected with HHV-6A, HHV-6B, and HHV-7 undergo morphological changes such as cell surface unevenness and hypertrophy. Among these, cells having irregularities on the cell surface are apoptotic cells because their diameter is the same as that of non-infected cells and in the case of apoptosis, the origin is a change in the cell membrane.
On the other hand, since hypertrophy of cells is a typical form of necrosis (reference document 24), hypertrophied cells are considered to be necrotic cells. In addition, although many cells showed a positive reaction by immunostaining, the number of cells that became apoptotic and necrotic was small, which revealed that many of the infected cells showed a normal state (morphology).
The above test revealed for the first time that cells infected with HHV-6A, HHV-6B, and HHV-7 induce not only apoptosis but also necrosis.
The above findings indicate that HHV-6A and HHV-6B, which are latently infected with nerve cells and glial cells in the hippocampus in which the neuron is newly formed in AD, undergo cell death including apoptosis of host cells. When reactivated, it also causes necrosis, and various inflammatory substances (eg, HMGB-1, TDP-43, GSK-3β, glutamic acid, Ca ++, etc.) from the cells as DAMPs (Damage/Danger Associated Molecular Patterns). Is released, suggesting enhanced inflammation.
（ｅ）安全性
　ホスカビル（登録商標）は、標的となるHHV-6A、HHV-6B及びHHV-7に対する有効量と毒性量とに大きな乖離があり（図３）、安全域が広い薬剤である。CMV感染症で長い間、日米欧等世界各国で承認を受け臨床使用されてきたことから（非特許文献１３）、安全にADでの治療に使用できる。 (E) Safety Hoscavir (registered trademark) is a drug with a wide safety margin, with a large difference between the effective dose and the toxic dose for the target HHV-6A, HHV-6B and HHV-7 (Fig. 3). .. Since it has been approved and clinically used in various countries around the world such as Japan, US and Europe for CMV infection (Non-patent Document 13), it can be safely used for treatment with AD.
（ｆ）ADへの治療効果
　本発明において、細胞外のAβはウイルスの有無に拘わらず細胞内に取り込まれることが確認されたことから（実施例１）、Aβがウイルス感染に対する自己免疫応答だとすれば、ウイルス感染を抑制しない限り、Aβの産生は止まらず、分泌されたAβは細胞内に取り込まれ蓄積する可能性が高い。AD発症にAβの病理作用が関与することから、Aβ産生の原因である脳内、特に海馬に潜伏しているHHV-6/７の再活性化を抑制することによるADの治療が期待できる。
　ADの主な罹患部位は海馬及びその周辺の神経組織であると考えられるが、この状況はHHV-6/7が海馬及びその周辺の神経細胞で再活性化し、そのことによりAβ蓄積、感染ウイルスや炎症部位に遊走したT細胞等による細胞傷害等の病理の発展による神経機能の崩壊過程であると説明できる（AD・「HHV-6脳炎カスケード仮説」）。そして、海馬の神経機能は再生能を持つので、死亡した細胞の補填や障害個所の修復が期待できる為、ホスカルネットなど本発明の抗HHV-6/7作用を有する抗ウイルス剤による早期治療を行えば、無症候性で済むことが期待できる。また、海馬歯状回等に存在する神経幹細胞層が崩壊していない限り、重度のADでも回復の可能性が見込まれる。
　感染部位に遊走した活性化T細胞は、通常、その役割を終えればアポトーシスにより死亡し何らの作用も起こさないが、本発明ではアポトーシス以外に、DAMPs等を放出するネクローシスも惹起することが確認された（実施例５）。これにより、AD発症の防止やその治療には、T細胞等の遊走をもたらす因子の排除が不可欠で、その因子と考えられるHHV-6/7の再活性化を抑制する抗ウイルス剤は適している。
　また、造血幹細胞や臓器の移植後に発症するHHV-6脳炎患者（「移植後HHV-6脳炎患者」）がADを発症した患者で観察される認知障害と類似した神経症状を示すことから、AD患者への抗HHV-6/7作用を有する抗ウイルス剤の最適な用法・用量としては、移植後HHV-6脳炎患者に対して有意な治療効果が確認された用法・用量が参考になる。例えば、ホスカルネットの場合、HHV-6脳炎患者に対し数日間のフルドーズ（180mg/kg/日）点滴静注投与の場合に優れた治療成績が報告されている。 (F) Therapeutic effect on AD In the present invention, it was confirmed that extracellular Aβ is taken up into cells regardless of the presence or absence of virus (Example 1). Therefore, Aβ is an autoimmune response to virus infection. Therefore, unless the viral infection is suppressed, the production of Aβ does not stop, and the secreted Aβ is likely to be taken up and accumulated in the cells. Since the pathological effect of Aβ is involved in the onset of AD, it can be expected to treat AD by suppressing the reactivation of HHV-6/7 latent in the brain, which is the cause of Aβ production, particularly in the hippocampus.
It is considered that the main affected area of AD is the neural tissue of the hippocampus and its surroundings. In this situation, HHV-6/7 is reactivated in the neurons of the hippocampus and its surroundings, which causes Aβ accumulation and infectious virus. It can be explained that it is a process of neuronal function disruption due to the development of pathology such as cell damage caused by T cells that have migrated to the inflammatory site (AD, "HHV-6 encephalitis cascade hypothesis"). And since the hippocampal nerve function has a regenerative ability, it can be expected to compensate for dead cells and repair damaged areas, so early treatment with an antiviral agent having the anti-HHV-6/7 action of the present invention such as foscarnet. If you do, you can expect to be asymptomatic. In addition, as long as the neural stem cell layer present in the dentate gyrus of the hippocampus is not destroyed, there is a possibility of recovery even with severe AD.
Activated T cells that have migrated to the site of infection usually die by apoptosis after finishing their role and cause no action.However, in the present invention, it was confirmed that in addition to apoptosis, necrosis that releases DAMPs etc. is also induced. (Example 5). Therefore, elimination of a factor that causes migration of T cells and the like is indispensable for prevention and treatment of AD onset, and an antiviral agent that suppresses reactivation of HHV-6/7 considered to be that factor is suitable. There is.
In addition, since HHV-6 encephalitis patients (“post-transplant HHV-6 encephalitis patients”) that develop after transplantation of hematopoietic stem cells or organs show neurological symptoms similar to the cognitive impairment observed in patients who developed AD, As an optimal dosage and administration of an antiviral agent having an anti-HHV-6/7 action to a patient, the dosage and administration confirmed to have a significant therapeutic effect on post-transplant HHV-6 encephalitis patients can be referred to. For example, in the case of foscarnet, excellent therapeutic results have been reported in patients with HHV-6 encephalitis when given by full-dose (180 mg/kg/day) intravenous infusion for several days.
　また、ホスカルネットにはCa＋＋と結合する作用があり、臨床的には低カルシウム血症などの副作用が知られている。反面、過剰になっている細胞の内外でのCa＋＋の低下をもたらす作用、恒常性を回復する作用（図５）が期待できる。特にAβはカルシウムチャネルとなり細胞内へ過剰なカルシウムの流入を高める作用があることから（A Drews et al., Scientific Reports 6:31910: 1-12, 2016）、Aβ産生の原因であるHHV-6/7の再活性化の抑制は、間接的に細胞内のCa＋＋の低下に寄与する。
 なお、ホスカルネットが脳内で再活性化したHHV-6/7に対する抗ウイルス作用を発揮するにはBBBを通過し感染部位に到達する必要がある。ホスカルネットはBBBを通過し、脳脊髄液中でHHV-6/7に効果を有する濃度を投与後6時間程度維持できる（図４）為、AD治療に適している。 Further, foscarnet has an action of binding to Ca ++, and clinically known side effects such as hypocalcemia. On the other hand, the action of reducing Ca ++ inside and outside the excessive cells and the action of restoring homeostasis (FIG. 5) can be expected. In particular, since Aβ acts as a calcium channel and enhances the influx of excess calcium into cells (A Drews et al., Scientific Reports 6:31910:1-12, 2016), HHV-6 which is the cause of Aβ production. Inhibition of /7 reactivation indirectly contributes to the reduction of intracellular Ca ++ .
In order for foscarnet to exert its antiviral effect on HHV-6/7 reactivated in the brain, it must pass through the BBB and reach the infection site. Foscarnet crosses the BBB and can maintain a concentration that has an effect on HHV-6/7 in cerebrospinal fluid for about 6 hours after administration (Fig. 4), and is therefore suitable for AD treatment.
　以上から、HHV-6/7に対する抗ウイルス剤の投与により、少なくともAβの生成、ヒト神経細胞内へのAβの取り込み、オリゴマーの形成、及び／又はAβの蓄積が抑制されるといえるから、ADはHHV-6/7に対して効果のある抗ウイルス剤によって治療が可能となる。
　HHV-6脳炎の主たるプレイヤーはHHV-6Bであるが、ADの場合は、疾患の拡大及び増悪にHHV-6A及びHHV-7の再活性化が関与している可能性が高く、更にHSV-1やサイトメガロウイルス（CMV）感染による脳炎においても認知症様の症状を呈することが知られていることから、これら他のヘルペスウイルスの再活性化もAβの分泌を促し、AD増悪の要因となる可能性もある。
　従って、AD治療を目指す抗ウイルス剤にはHHV-6BのみならずHHV-6A及びHHV-7に対する作用も、更にはHSV-1やCMVなど他のヘルペスウイルスに対するマルチ抗ウイルス作用を持つことが望ましい。ホスカルネットは、このようなマルチ抗ウイルス活性を有する為、ADへの治療効果が期待できる。 From the above, it can be said that administration of an antiviral agent against HHV-6/7 suppresses at least Aβ production, Aβ uptake into human nerve cells, oligomer formation, and/or Aβ accumulation. Can be treated with antiviral agents effective against HHV-6/7.
The main player of HHV-6 encephalitis is HHV-6B, but in the case of AD, it is highly possible that reactivation of HHV-6A and HHV-7 is involved in the spread and exacerbation of the disease, and further HSV- It is known that encephalitis caused by 1 and cytomegalovirus (CMV) infection also exhibits dementia-like symptoms. Therefore, reactivation of these other herpesviruses also promotes secretion of Aβ, which is a factor of AD exacerbation. There is also a possibility.
Therefore, it is desirable that an antiviral agent aiming at AD treatment has an action not only on HHV-6B but also on HHV-6A and HHV-7, and further has a multi-antiviral action against other herpesviruses such as HSV-1 and CMV. .. Since foscarnet has such multi-antiviral activity, it can be expected to have a therapeutic effect on AD.
　＜本発明の抗ウイルス剤のADの治療及び予防用途にける実施の態様＞
　本発明は、ヒトヘルペスウイルス6型（HHV-6A及びHHV-6B）及び7型（HHV-7）等に対し、優れた抗ウイルス作用を有する抗ウイルス剤をADの治療及び予防に用いるという医薬用途に関し、以下の第１ないし第１１の実施形態を含むが、それらに限定されない。 <Aspects of Implementation of Antiviral Agent of the Present Invention for Treatment and Prevention of AD>
The present invention relates to a human herpesvirus type 6 (HHV-6A and HHV-6B) and type 7 (HHV-7) and the like, wherein an antiviral agent having an excellent antiviral action is used for treating and preventing AD. Applications include, but are not limited to, the following first through eleventh embodiments.
　第１の形態は、アルツハイマー病（AD）を治療（予防を含む）に用いることのできる抗ウイルス剤は、ヒトヘルペスウイルス６型（HHV-6A及び／又はHHV-6B）及び／又はヒトヘルペスイルス７型（HHV-7に対する強い抗ウイルス作用を有する化合物を有効成分とする薬剤であって、望ましくは単純ヘルペスウイルス1型（HSV-1）、サイトメガロウイルス（CMV）などに対しても効果のあるマルチ抗ウイルス作用を有する化合物を有効成分とする薬剤である。 In the first form, an antiviral agent that can be used for treatment (including prevention) of Alzheimer's disease (AD) is human herpesvirus 6 (HHV-6A and/or HHV-6B) and/or human herpesvirus. Type 7 (A drug containing a compound having a strong antiviral action against HHV-7 as an active ingredient, and is preferably effective against herpes simplex virus type 1 (HSV-1), cytomegalovirus (CMV), etc. It is a drug containing a compound having a multi-antiviral action as an active ingredient.
　第２の形態は、上記抗ウイルス作用を有する化合物には、ヘルペスウイルスのDNAポリメラーゼを阻害する化合物、ウイルスDANの合成に必須のヘリカーゼ・プライマーゼ複合化合物、ウイルスDNAのカプシドへのパッケージ阻害化合物及びウイルスDNAポリメラーゼのピロリン酸と直接結合してDNA合成を選択的に阻害する化合物、ピロリン酸アナログがある。その内、ピロリン酸アナログ（リン酸類縁化合物）はリン酸化酵素による活性化を必要とせず、耐性の出る可能性は低く、長期投与が予定されているAD治療に適している。
　ピロリン酸アナログ以外の抗ウイルス作用を有する化合物も選択的にウイルスDNAポリメラーゼを阻害するなどウイルスの増殖の阻害作用が期待できる。特にピロリン酸アナログと併用することで、ピロリン酸アナログの抗ウイルス効果を補完することができる。 In the second embodiment, the compound having the above-mentioned antiviral activity includes a compound that inhibits the DNA polymerase of herpesvirus, a helicase-primase complex compound essential for the synthesis of viral DAN, a compound that inhibits the packaging of viral DNA into a capsid, and There is a pyrophosphate analog, a compound that directly binds to pyrophosphate of viral DNA polymerase and selectively inhibits DNA synthesis. Of these, pyrophosphate analogs (phosphate analogs) do not require activation by phosphatase, are unlikely to develop resistance, and are suitable for AD treatment for long-term administration.
Compounds having an antiviral effect other than the pyrophosphate analog can be expected to have an inhibitory effect on virus growth, such as selectively inhibiting viral DNA polymerase. In particular, when used in combination with the pyrophosphate analog, the antiviral effect of the pyrophosphate analog can be complemented.
　第３の形態は、上記ピロリン酸アナログが、ホスホノ酢酸もしくはホスホノギ酸及び／又はそれらの誘導体であり、DNAポリメラーゼのピロリン酸結合部位に結合し、選択的にウイルス増殖を阻害する活性や、カルシウム拮抗作用及び抗アスパラギン酸プロテアーゼ作用が期待できる。なお、下記基本骨格（式１）で示されるホスホノギ酸もしくはその塩、又はこれらの溶媒和物を「ホスホノギ酸誘導体」という。 In a third form, the above-mentioned pyrophosphate analog is phosphonoacetic acid or phosphonoformic acid and/or a derivative thereof, which binds to the pyrophosphate binding site of DNA polymerase and selectively inhibits virus growth, and calcium antagonism. The action and anti-aspartic protease action can be expected. The phosphonoformic acid represented by the following basic skeleton (formula 1) or a salt thereof, or a solvate thereof is referred to as a “phosphonoformic acid derivative”.
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
第４の形態が、ホスホノギ酸誘導体であり、分子式が「CNa3O5P・6H2O」であって以下の（式２）示される化合物。本発明に限定するものではないが、本実施例では「ホスカルネット」として、（式２）で示される化合物を用いている。 A fourth form is a phosphonoformic acid derivative, which has a molecular formula of “CNa 3 O 5 P·6H 2 O” and is represented by the following (formula 2). Although not limited to the present invention, the compound represented by (Formula 2) is used as the "foscarnet" in this example.
Figure JPOXMLDOC01-appb-C000009
 （式２）
Figure JPOXMLDOC01-appb-C000009
 (Formula 2)
　上記ホスカルネットの製剤（注射剤）は「ホスカビル（登録商標）」（日本）あるいは「Foscavir」（米国等）の商品名で、主にサイトメガロウイルス感染症の適応症で世界各国で市販されている。 The above foscarnet preparation (injection) is sold under the trade name of "Foscavir (registered trademark)" (Japan) or "Foscavir" (US etc.) and is mainly marketed worldwide for indication of cytomegalovirus infection. ing.
　第５の形態は、ホスカルネットを有効成分とする製剤及び経口製剤をADの治療（予防を含む）の為に使用する方法。
　この使用には、Preclinical ADの治療を含む。Preclinical ADとは、脳にアミロイドβの蓄積が証明された段階から初期認知障害（MCI）の前までの状態をいい、Stage 1は脳内にアミロイドβの蓄積がみられる段階、Stage 2 はアミロイドβの蓄積に脳の変性所見が伴う段階、Stage 3はStage 2に加えてごくわずかな認知機能の低下が始まった段階の3段階からなる。Preclinical ADに対する治療では、望ましくはStage 3、更に望ましくはStage 2の段階で開始する。 The fifth mode is a method of using a formulation containing foscarnet as an active ingredient and an oral formulation for the treatment (including prevention) of AD.
This use includes treatment of Preclinical AD. Preclinical AD refers to the state from the stage where the accumulation of amyloid β in the brain was proved to the stage before the initial cognitive impairment (MCI), Stage 1 was the stage where amyloid β was accumulated in the brain, and Stage 2 was the amyloid. In addition to Stage 2, Stage 3 consists of three stages: the accumulation of β accompanied by degenerative findings in the brain, and the stage where a slight decrease in cognitive function began. Treatment for Preclinical AD preferably begins at Stage 3, more preferably Stage 2.
　第６の形態は、AD治療の為のホスカルネットの製剤が注射剤で、その使用が先制療法をも目的とする方法。先制療法は、脳脊髄液中及び／又は血漿もしくは血液中のHHV-6A、HHV-6B及びHHV-7の各ウイルスDNA量を有意に低下させる。望ましくは健常人レベル以下、更に望ましくは検知水準化以下に低下させることを目的として、AD患者を入院させ、例えば、ホスカルネットの先制療法の有効量（60～180mg/kg/日）を、1日2回又は3回に分けて、髄腔内投与あるいは点滴静注により3～10日間、望ましくは3ないし5日間、継続して投与する方法。
　尚、各ウイルスのDNAを検査する方法は、非特許文献３）に定める方法、その他医療機関が通常利用できる方法でよい。尚、低下目標となる各ウイルスDNA量は患者の状態を勘案し、適宜修正可能である。
　また、各ウイルスのDNA量の減少及びその維持状況、患者の認知機能の回復の状況を勘案し、並びにホスカルネットの忍容性の維持又は腎障害などの重篤な副作用の発生を防止もしくはコントロールする為、先制療法の期間及びホスカルネットの1回当たり又は1日当たりの投与量を適宜調整することができる。また、髄腔内投与については、薬剤が直接感染部位に移行できるため、投与量を更に低減することができる。 The sixth form is a method in which a foscarnet preparation for treating AD is an injection, and the use thereof is also a preemptive therapy. Preemptive therapy significantly reduces the amount of HHV-6A, HHV-6B and HHV-7 viral DNA in cerebrospinal fluid and/or plasma or blood. For the purpose of lowering the level below the level of healthy subjects, more preferably below the level of detection, AD patients are admitted to the hospital, and, for example, the effective dose (60-180 mg/kg/day) of pretreatment with foscarnet is A method of continuous administration by intrathecal administration or intravenous drip infusion for 3 to 10 days, preferably 3 to 5 days, divided into 2 or 3 times a day.
The method for inspecting the DNA of each virus may be the method defined in Non-Patent Document 3) or any other method commonly used by medical institutions. In addition, the amount of each viral DNA, which is a reduction target, can be appropriately modified in consideration of the patient's condition.
In addition, in consideration of the decrease in DNA amount of each virus and its maintenance status, the situation of recovery of cognitive function of patients, and prevention of serious side effects such as maintenance of tolerability of foscarnet or renal damage, or For control, the duration of preemptive therapy and the dose of foscarnet per day or per day can be adjusted appropriately. Further, for intrathecal administration, the drug can be directly transferred to the infected site, and thus the dose can be further reduced.
　第７の形態は、ホスカルネットの製剤が注射剤で、先制療法の後の維持療法として、各ウイルスの再活性化の防止を目的とし、例えば、ホスカルネットの有効量として60～120mg/kg/日を、1日、2ないし3回に分けて、静注もしくは筋注もしくは皮下注で、望ましくは点滴静注で、3ないし6ヵ月間、投与する方法。
　なお、各ウイルスDNAの減少及びその維持状況、患者の認知機能の回復の状況を勘案し、並びにホスカルネットの忍容性の維持又は腎障害などの重篤な副作用の発生を防止もしくはコントロールする為、維持療法の期間並びにホスカルネットの1回当たり又は1日当たりの投与量を適宜調整することができる。 The seventh form is an injectable foscarnet preparation, which is a maintenance therapy after pre-emptive therapy for the purpose of preventing reactivation of each virus. For example, an effective dose of foscarnet is 60 to 120 mg/ A method in which kg/day is divided into 2 to 3 times a day and administered intravenously, intramuscularly or subcutaneously, preferably intravenous drip for 3 to 6 months.
In addition, in consideration of the reduction and maintenance status of each viral DNA, the status of recovery of cognitive function in patients, and to prevent or control the occurrence of serious side effects such as maintenance of tolerability of foscarnet or renal damage. Therefore, the duration of maintenance therapy and the dose of foscarnet per dose or per day can be appropriately adjusted.
　第８の形態は、ホスカルネットの製剤が経口剤で、注射剤による維持療法と共に又はこれに代替して、例えば、ホスカルネットの1日当たり有効量として2000mgないし6000mgを、1日に3ないし5回に分けて、3ないし6ヵ月間、継続して投与することを特徴とする維持治療の方法。ホスカルネットは粘膜刺激性を有することから、その経口剤の投与に際しては、テプレノン、スクラルファート、アズレンスルフォル酸ナトリウム、レパミピド、ポラプレジンク、イルソグラジンマイレン酸塩、ベキサネート塩酸塩、ソファルコン、セトラキサート塩酸塩、エカベトナトリウム水和物等の胃粘膜保護作用のある薬剤との併用あるいはそれらとの合剤が望ましい。
　なお、各ウイルスDNAの減少及びその維持状況、患者の認知機能の回復の状況を勘案し、並びにホスカルネットの忍容性の維持又は腎障害などの重篤な副作用の発生を防止もしくはコントロールする為、維持療法の期間並びにホスカルネットの1回当たり又は1日当たりの投与量を適宜調整することができる。 In an eighth form, the foscarnet preparation is an oral preparation, and with or as an alternative to maintenance therapy by injection, for example, the effective daily dose of foscarnet is 2000 mg to 6000 mg, or 3 to 1 day. A method of maintenance treatment, characterized by continuous administration for 3 to 6 months in 5 divided doses. Since foscarnet has mucosal stimulant properties, teprenone, sucralfate, sodium azulensulforate, repamipide, polaprezinc, irsogladine maleate, bexanate hydrochloride, spartalcon, and cetraxate hydrochloride are administered when its oral preparation is administered. A combination with a drug having a gastric mucosa-protecting action such as salt or ecabet sodium hydrate, or a combination thereof is preferable.
In addition, in consideration of the reduction and maintenance status of each viral DNA, the status of recovery of cognitive function in patients, and to prevent or control the occurrence of serious side effects such as maintenance of tolerability of foscarnet or renal damage. Therefore, the duration of maintenance therapy and the dose of foscarnet per dose or per day can be appropriately adjusted.
　第９の形態は、ADを治療（予防を含む）する為の、ホスカルネットの製剤と化学的に又は作用メカニズムの異なる抗ウイルス作用を有する化合物、もしくは当該化合物を含有する製剤とを併用して使用する方法。
　併用する抗ウイルス剤には、ファムシクロビル（特開平０４－２７５２２９号）、アシクロビル（米国特許第４９５７９２４号）、バラシクロビル（特許第３３５００５５号）、バルガンシクロビル、ペンシクロビル、ブリブジンなどのヌクレオシド類縁体を含有する製剤、シドフォビル（特許第５９６３７８７号）、そのプロドラッグもしく誘導体などのヌクレオタイド類縁体を含有する製剤、又はヌクレオシド化合物を含有する製剤、非ヌクレオシドDNAポリメラーゼ抑制化合物を含有する製剤、ヘリカーゼ／プライマーゼ複合体抑制化合物を含有する製剤、ウイルスDNAカプシドへのパッケージ阻害化合物、抗CD40抗体（特表２００２－５４３１５０号）、PD-1抗体（特許第５７０１２６６号）、テトラヒドロー２Ｈ－チオピランーカルボキサミド誘導体（特許第５０１１７３９号）、グレリン誘導体（特開２０１８－１３８５８６号）を含有する製剤があるが、これらに限定されない。 The ninth mode is a combination of a foscarnet preparation and a compound having an antiviral effect having a different chemical or mechanism of action, or a preparation containing the compound, for treating (including preventing) AD. How to use.
The antiviral agent used in combination contains nucleoside analogs such as famciclovir (JP 04-275229A), acyclovir (US Pat. No. 4,957,924), valacyclovir (patent 3350055), valganciclovir, penciclovir and brivudine. Formulation, cidofovir (Patent No. 5963787), a formulation containing a nucleotide analog such as a prodrug or derivative thereof, a formulation containing a nucleoside compound, a formulation containing a non-nucleoside DNA polymerase inhibitor compound, helicase/primer Preparation containing ze complex inhibiting compound, compound inhibiting package to viral DNA capsid, anti-CD40 antibody (Japanese Patent Publication No. 2002-543150), PD-1 antibody (Patent No. 5701266), tetrahydro-2H-thiopyran-carboxamide derivative (Patent No. 5011739) and a preparation containing a ghrelin derivative (JP-A-2018-138586), but the preparation is not limited thereto.
　第１０の形態は、ADを治療及び予防する為、ホスカルネット製剤を、作用メカニズムの異なる他のADの治療を目的とする薬剤（AD治療剤）、具体的にはADの悪化要因となるAβの凝集、タウタンパク質のリン酸化／神経原線維変化抑制又はアセチルコリンなどの神経伝達物質の生成・伝達の調製に直接的な作用を有する化合物、もしくは当該化合物を有効成分とする製剤と併用する方法。
　ホスカルネットは、過剰な細胞死の原因ウイルスの増殖を抑えることで、自然免疫反応としての過剰なAβの産生や分泌を抑制する作用がある。
　また、ホスカルネットが細胞内で抗アスパラギン酸プロテアーゼ作用を発揮する可能性があり、Aβの生成を抑制することが期待できる。ただ、強い作用は得られていない為、ADの治療に当たっては、Aβの凝集やタウタンパク質のリン酸化や神経原線維変化の抑制、又はアセチルコリンなどの神経伝達物質の生成・伝達の調製に直接的な作用を有する化合物、もしくは当該化合物を有効成分とする薬剤との併用は有用である。
　併用の対象となる薬剤としては、ドネペジルのようなアセチルコリンエステラーゼを阻害する薬剤あるいはガランタミンのようなアセチルコリンエステラーゼ阻害と共にニコチン酸受容体増強作用を有する薬剤、リバスチグミンのようなアセチルコリンエステラーゼとButyrylcholine Esteraseの双方を阻害する薬剤、メマンチンのようなNMD受容体を阻害する薬剤がある。
　更に、Aβを分解するセクレターゼに作用してAβの生成を阻害する薬剤（特表２０１０－５２９０１４、特表２０１１－５１８２２４）、又はAβの蓄積を阻害しあるいは分解を促進する薬剤、又はAβの脳室内からの排出を促進する薬剤、又はAβの凝集あるいはAβプロトフィブリルの生成を阻害する薬剤（特表２０１７－５２１４２７）、又はタンパク質の重合・活性化抑制剤（特表２００３－５２８１７１、特表２０１７－５２１４２７）又は酸化ストレス／小胞体ストレス誘導アポトーシス抑制剤（特開２００８－２３９５３８）、ワクチン（特開２００７－５２２１１９、特表２０１０－５２２５５９）などとの併用も可能である。
　更に、細胞への過剰なカルシウム流入抑制剤、イブプロフェン、ギンコエキス、もしくは他の抗炎症剤、インスリンもしくは他の抗糖尿病薬、抗IL-6抗体、抗IL-12抗体、もしくは他の炎症性サイトカイン、ケモカインの中和する薬剤との併用も可能である。 In the tenth form, a foscarnet preparation is used for treating and preventing AD, and is a drug for treating other AD having a different mechanism of action (AD therapeutic agent), specifically, a factor that worsens AD. Method of combining with Aβ aggregation, phosphorylation of tau protein/inhibition of neurofibrillary tangle or production/transmission of neurotransmitters such as acetylcholine, or a compound containing the compound as an active ingredient ..
Foscarnet has an action of suppressing excessive Aβ production and secretion as an innate immune reaction by suppressing the proliferation of virus causing excessive cell death.
In addition, foscarnet may exert an anti-aspartic protease action in cells, and it can be expected to suppress the production of Aβ. However, since a strong action has not been obtained, in treating AD, direct inhibition of Aβ aggregation, phosphorylation of tau protein and neurofibrillary tangles, or preparation of production/transmission of neurotransmitters such as acetylcholine. It is useful to use a compound having various actions or a drug containing the compound as an active ingredient.
Drugs that can be used in combination include drugs that inhibit acetylcholinesterase such as donepezil, drugs that have a nicotinic acid receptor-enhancing effect with acetylcholinesterase inhibition such as galantamine, and both acetylcholinesterase and butyrylcholine esterase such as rivastigmine. There are drugs that block, drugs that block NMD receptors such as memantine.
Furthermore, agents that act on secretase that decomposes Aβ to inhibit the production of Aβ (Tables 2010-5209014 and 2011-518224), agents that inhibit the accumulation of Aβ or promote the degradation, or the brain of Aβ Agents that promote excretion from the room, agents that inhibit Aβ aggregation or Aβ protofibril formation (Table 2017-521427), or inhibitors of protein polymerization/activation (Table 2003-528171, Table 2017) -521427), an oxidative stress/endoplasmic reticulum stress-induced apoptosis inhibitor (JP-A 2008-239538), a vaccine (JP-A 2007-522119, JP 2010-522559), and the like can also be used in combination.
Furthermore, an excessive calcium influx inhibitor into cells, ibuprofen, ginkgo extract, or other anti-inflammatory drug, insulin or other antidiabetic drug, anti-IL-6 antibody, anti-IL-12 antibody, or other inflammatory cytokine It can also be used in combination with a drug that neutralizes chemokines.
　第１１の形態は、ADの診断及び治療薬の効果を確認するバイオマーカーとして、AD患者のHHV-6A及びHHV-6B並びにHHV-7のウイルス自体、ウイルスDNA又は抗体もしくはその断片を、唾液、血液、血漿、脳脊髄液（CSF）などから検出する方法である。その中で、脳、特に海馬でのウイルス感染を確認する方法として最も精度が高いのはCSF中のウイルスDNAを測る方法である。
　その際、ADに罹患するリスクのある人もしくはADの進展状況の診断及びADの治療に用いる抗ウイルス剤の治療効果を確認するバイオマーカーとして、被験者の脳脊髄液中もしくは血漿中もしくは血液中、もしくは唾液中、望ましくは血液中もしくは唾液中のHHV-6A及びHHV-6B並びにHHV-7のそれぞれに特異的なウイルスDNA、蛋白質、糖脂質、あるいはウイルス組成グリコプロテインなどを用いる。この場合、アミロイドPET、タウPET、MRIなどADに関する診断方法やバイオマーカーと併用することができる。
　ADに罹患するリスク診断、ADの進展状況の診断及びADの治療に用いる抗ウイルス剤を含むAD治療薬の治療効果を確認する方法のバイオマーカーとしてのCSF中のアミロイド、タウタンパク質、アミロイドPET検査、フルオロ－D－グルコースPET、MRI検査が汎用され、また、ADの診断及び治療効果を確認する為のバイオマーカーとして、多くの方法が開発されている。
　しかし、HHV-6A、HHV-6B及びHHV-7のウイルスDNAをAD診断のバイオマーカーに用いる方法は知られていない。
　ウイルスDNAを測る方法に関する新規な技術は開発されているが、国立感染症研究所が作成している突発性発疹の原因ウイルスであるHHV-6及びHHV-7に関する「病原体検査マニュアル」（参考文献２４）に定めるリアルタイムPCR法が標準的な方法として利用できる In the eleventh form, HHV-6A and HHV-6B of AD patients and HHV-7 virus as a biomarker for confirming the effect of a drug for diagnosing AD and therapeutic agents, viral DNA or antibody or a fragment thereof, saliva, It is a method to detect from blood, plasma, cerebrospinal fluid (CSF), etc. Among them, the most accurate method for confirming viral infection in the brain, especially the hippocampus, is to measure viral DNA in CSF.
At that time, as a biomarker for confirming the therapeutic effect of an antiviral agent used for the diagnosis of AD at the risk of suffering from AD or the development of AD, in the cerebrospinal fluid or plasma or blood of the subject, Alternatively, viral DNA, protein, glycolipid, or viral composition glycoprotein specific to HHV-6A and HHV-6B and HHV-7 in saliva, preferably in blood or saliva are used. In this case, it can be used in combination with a diagnostic method for AD or a biomarker such as amyloid PET, tau PET, and MRI.
Amyloid in CSF, tau protein, amyloid PET test as a biomarker of a method for diagnosing the risk of suffering from AD, diagnosing the progress of AD, and confirming the therapeutic effect of an AD therapeutic drug including an antiviral agent used for AD treatment , Fluoro-D-glucose PET, MRI examination are widely used, and many methods have been developed as biomarkers for confirming the diagnostic and therapeutic effects of AD.
However, a method of using the viral DNAs of HHV-6A, HHV-6B and HHV-7 as a biomarker for AD diagnosis is not known.
Although a new technique for measuring viral DNA has been developed, the "Pathogen Inspection Manual" (HBV-6 and HHV-7), which are the causative viruses of exanthema subitum, is being prepared by the National Institute of Infectious Diseases (Reference) The real-time PCR method specified in 24) can be used as a standard method.
　本発明に係るホスカルネットの製剤は、ADの治療だけでなく、潜伏感染しているヘルペスウイルス類、特にHHV-6及びHHV-7の再活性化及び／又はその増殖、並びにアミロイドβの蓄積、及びホスカルネットの抗ウイルス作用が効果を有するウイルス感染が原因とする以下の疾患の治療剤としても使用可能である。
　HHV-6脳炎（乳幼児）、突発性発疹、肺炎、加齢黄斑変性症、口腔癌、慢性疲労症候群、大うつ症候群、双極性障害、心不全、薬物過敏症、特発性血小板減少紫斑症、バラ色粃糠疹、多発性硬化症、レビー小体変性症、脊髄・小脳変性症、髄膜炎、橋本病、癲癇、内側側頭葉硬化症、前頭側頭葉変性症、筋委縮側索硬化症、ハンチントン病、ラスムッセン脳炎、自己免疫性脳炎、辺縁系脳炎、HSV-1性脳炎、CMV性脳炎、HIV性脳炎、EVB性脳炎、EKV腎症、造血幹細胞・骨髄あるいは臓器移植後の出血性膀胱炎・間質性腎炎・尿管狭窄を治療（予防を含む）する為の使用。 The preparation of foscarnet according to the present invention not only treats AD but also reactivates latently infected herpesviruses, particularly HHV-6 and HHV-7, and/or their proliferation, and accumulation of amyloid β. , And the antiviral effect of foscarnet can be used as a therapeutic agent for the following diseases caused by viral infection.
HHV-6 encephalitis (infant), idiopathic rash, pneumonia, age-related macular degeneration, oral cancer, chronic fatigue syndrome, major depression syndrome, bipolar disorder, heart failure, drug hypersensitivity, idiopathic thrombocytopenic purpura, rosy color Pityriasis, multiple sclerosis, Lewy body degeneration, spinal and cerebellar degeneration, meningitis, Hashimoto's disease, epilepsy, medial temporal lobe sclerosis, frontotemporal lobar degeneration, muscle atrophy lateral sclerosis , Huntington's disease, Rasmussen encephalitis, autoimmune encephalitis, limbic encephalitis, HSV-1 encephalitis, CMV encephalitis, HIV encephalitis, EVB encephalitis, EKV nephropathy, hematopoietic stem cell/bone marrow or hemorrhagic after organ transplantation Use to treat (including preventive) cystitis, interstitial nephritis, ureteral stenosis.
　本発明に係るホスカルネットの非経口の製剤として、皮下、筋肉内、髄腔内又は静脈内投与用の注射剤、点滴剤、点滴用パック剤、経鼻用などの剤型がある。
　先制療法用として望ましいのは高用量の投与が可能な静脈注射用に調製された製剤、更に望ましいのは点滴静注用の製剤である。注射剤の場合、当事業者で公知の方法、適当な希釈剤（生理食塩水など）にホスカルネットを溶解し、過滅菌等の滅菌処理を施し、アンプル、バイアル、輸液パックなどの密封容器に充填することにより製造できる。 The parenteral preparation of foscarnet according to the present invention includes dosage forms for subcutaneous, intramuscular, intrathecal or intravenous administration, infusions, drip packs, nasal administration and the like.
Desirable for preemptive therapy is a formulation prepared for intravenous injection capable of high dose administration, and more desirable is a formulation for intravenous drip infusion. In the case of injections, foscarnet is dissolved in a suitable diluent (physiological saline, etc.) known to those skilled in the art, sterilized by oversterilization, etc., and sealed containers such as ampoules, vials, infusion packs, etc. It can be manufactured by filling.
　維持療法に用いる経口用のホスカルネット単独製剤の場合、製剤は一般的な製造方法、賦形剤、崩壊剤、結合剤、滑沢剤、懸濁化剤、等張化剤、乳化剤、甘味料、香料、着色料などの添加剤とホスカルネットを常法により混合することにより製造できる。
　ホスカルネットは極めて安定性の高い化合物であるが金属類をキレート形成しやすいこと、粘液刺激性があることから、賦形剤としてはデンプン、乳糖、マニトールなど容易に分解しやすいものが望ましい。
　また、腸管からの吸収を早め、又は徐放の為に、公知の方法でマイクロカプセルにホスカルネットを封入し、又は多重構造の錠剤とすることができる。
　ホスカルネットとして1グラムを超えるような高用量の製剤を用いる場合、通常の錠剤やカルセル（ソフトカプセルを含む）では製剤が大きく高齢者の服用は難しくなる。そこで、顆粒剤、細粒剤、粉剤やゼリー製剤も利用できる。
　ドリンクなどの経口液剤とする場合、基剤としては注射用蒸留水あるいは生理食塩水を用い、ホスカルネットを常法に従って溶解し、必要に応じて食塩又は糖、シロップ等で加味し、ガラス製のミニボトルやPET容器などに充填することで製造できる。
　また、ホスカルネットは粘膜刺激性を有することから、その経口剤の投与に際しては、テプレノン、スクラルファート、アズレンスルフォル酸ナトリウム、レパミピド、ポラプレジンク、イルソグラジンマイレン酸塩、ベキサネート塩酸塩、ソファルコン、セトラキサート塩酸塩、エカベトナトリウム水和物等の胃粘膜保護作用のある薬剤との併用あるいはそれらとの合剤が望ましい。 In the case of an oral foscarnet single preparation used for maintenance therapy, the preparation is prepared by a general manufacturing method, excipient, disintegrant, binder, lubricant, suspending agent, tonicity agent, emulsifier, sweetness. It can be produced by mixing foscarnet with an additive such as a coloring agent, a fragrance, or a coloring agent by a conventional method.
Foscarnet is a compound with extremely high stability, but it is preferable to use an easily decomposing substance such as starch, lactose, or mannitol as an excipient because it easily chelates metals and has a mucous stimulating property.
Further, in order to accelerate absorption from the intestinal tract or to gradually release it, foscarnet can be encapsulated in microcapsules by a known method, or tablets having a multi-structure can be formed.
When using a high-dose formulation that exceeds 1 gram as foscarnet, conventional tablets and carcasses (including soft capsules) are large in formulation and difficult for the elderly to take. Therefore, granules, fine granules, powders and jelly preparations can also be used.
When used as an oral liquid such as a drink, distilled water for injection or physiological saline is used as a base, foscarnet is dissolved according to a conventional method, and salt or sugar, syrup, etc. are added if necessary, and it is made of glass. It can be manufactured by filling a mini bottle or PET container of.
In addition, since foscarnet has mucosal stimulant properties, teprenone, sucralfate, sodium azulensulforate, repamipide, polaprezinc, irsogladine maleate, bexanate hydrochloride, sofarcone, when administering its oral preparation, A combination with a drug having a gastric mucosal protective action such as cetraxate hydrochloride, ecabet sodium hydrate or the like or a combination thereof is desirable.
　ホスカルネットとAD剤あるいは胃粘膜保護剤との合剤は、ホスカルネットの単剤と同様な方法で製造できる。このほかにホスカルネットを封入したマイクロカプセルとAD剤あるいは胃粘膜保護剤を封入したマイクロカプセル剤（ナノチューブ・カプセルを含む）を混合することでも製造できる。 A mixture of foscarnet and AD agent or gastric mucosa protective agent can be produced in the same manner as foscarnet single agent. Alternatively, it can be produced by mixing microcapsules encapsulating foscarnet with microcapsules (including nanotube capsules) encapsulating AD agent or gastric mucosa protective agent.
　ADの診断に関しては、症状に応じて初期（軽度）、中期（中程度）、後期（高度）の３段階に分けられており、既存のAD治療薬もこの段階に応じた適応を取得している。
　ホスカルネットは、作用メカニズム的にはADのいずれの段階においても使用可能である。点滴靜注という推奨される用法用量からは重度ADの治療に適した薬剤であるが、HHV-6脳炎に対する治療経験からは、初期あるいは軽度の段階での投与がADの進行を止め、回復が期待できるので望ましい。このことは、ホスカルネットのPreclinical ADに対する利用を可能にする。
　Preclinical ADとは、脳にAβの蓄積が証明された段階から初期認知障害（MCI）の前までの状態をいい、stage 1は脳内にAβの蓄積がみられる段階、 stage 2 はAβの蓄積に脳の変性所見を伴う段階、 stage 3はstage 2に加えてごくわずかな認知機能の低下が始まった段階の3段階からなる。Preclinical ADに対する治療では、望ましくはstage 3 更に望ましくはstage 2 の段階で開始するとされているが、ホスカルネットは、この時点での治療にも適している。 Diagnosis of AD is divided into three stages, initial (mild), intermediate (medium), and late (advanced) according to the symptoms, and existing AD treatments have also acquired indications according to this stage. There is.
Foscarnet can be used at any stage of AD in terms of mechanism of action. From the recommended dosage of drip infusion, it is a drug suitable for treatment of severe AD, but from the experience of treatment for HHV-6 encephalitis, administration at an early or mild stage stops AD progression and recovery. It is desirable because it can be expected. This allows the use of foscarnet for Preclinical AD.
Preclinical AD refers to the state from the stage where Aβ accumulation is proved in the brain to the stage before early cognitive impairment (MCI). Stage 1 is the stage where Aβ accumulation is observed in the brain, and stage 2 is Aβ accumulation In addition to stage 2, stage 3 is accompanied by degenerative findings in the brain, and in addition to stage 2, a slight decline in cognitive function begins. Although treatment for Preclinical AD is preferably initiated at stage 3, more preferably stage 2, foscarnet is also suitable for treatment at this time.
　ADに関するバイオマーカーとして、脳脊髄液（CSF）中のAβ及びAβ42と他のAβとの比率、タウタンパク質、アミロイドPET検査、フルオロ－D－グルコースPET、MRI検査などが汎用されている。しかし、これらの方法では、ADの主因である感染ウイルスの有無や種類を特定できない。
　HHV-6A及びHHV-6B並びにHHV-7のウイルスDNAを測る方法に関する技術進化は目覚ましいが、国立感染症研究所が作成している突発性発疹の原因ウイルスであるHHV-6及びHHV-7に関する「病原体検査マニュアル」（参考文献２４）に定めるリアルタイムPCR法が標準的な方法として利用できる。 As biomarkers for AD, the ratios of Aβ and Aβ 42 to other Aβ in cerebrospinal fluid (CSF), tau protein, amyloid PET test, fluoro-D-glucose PET, MRI test, etc. are widely used. However, these methods cannot identify the presence or type of infectious virus that is the main cause of AD.
Although the technological evolution of the method for measuring viral DNA of HHV-6A, HHV-6B and HHV-7 is remarkable, it is related to HHV-6 and HHV-7 which are the causative viruses of exanthema subitum created by the National Institute of Infectious Diseases. The real-time PCR method defined in "Pathogen Inspection Manual" (reference document 24) can be used as a standard method.
　本発明では潜伏HHV-6/7の再活性化の抑制によりADを治療しようとするものであり、脳内のHHV-6/7のDNAレベルを比較的低侵襲的に測定可能な脳脊髄液（CSF）に着目し最も感度の高い検査方法であるリアルタイムPCR法で測定する。
　ADに対する抗ウイルス剤療法ではCSF中のHHV-6/7のDNAレベルを予防・治療効果の指標として扱う。
　AD患者のCSF中のHHV-6A及びHHV-6B、並びにHHV-7のウイルスDNAを検出限界以下とすることが最も望ましい。AD患者の脳内（海馬傍回など）のHHV-6A及びHHV-7のDNA量が非AD者の約2倍検出された旨の報告があることから、少なくとも非AD患者レベル以下のDNA量にすることが望ましい。
　急性の重篤な症状を示すHHV-6脳炎に対する先制療法として、ホスカルネットを数日間（最短で3日）最高用量（180mg/kg/日）での投与で脳脊髄液中のHHV-6BのDNAを検出限界以下にできたという報告がある。
　しかし、ADの場合は、確かに認知機構低下症状としてはHHV-6脳炎と共通するものの、HHV-6/7の高濃度感染・高活性のHHV-6脳炎とは異なり、穏やかに進行すると考えられている。従って、AD治療におけるホスカルネットの用法用量も、かなり穏やかな設定にできる可能性が高い。
　HHV-6の潜伏形態から、その再活性化を完全に抑制することはできない。先制療養で健常人レベルあるいは脳脊髄液中のウイルスDNAが検出限界以下にした後、維持療法に切り替え、患者のコンプライアンスを高める用法用量と製剤にする方法も選択できる。 In the present invention, it is intended to treat AD by suppressing the reactivation of latent HHV-6/7, and the cerebrospinal fluid capable of measuring the DNA level of HHV-6/7 in the brain in a relatively minimally invasive manner. Focus on (CSF) and measure by the most sensitive test method, real-time PCR.
In antiviral drug therapy for AD, the DNA level of HHV-6/7 in CSF is treated as an index of preventive and therapeutic effects.
Most preferably, the HHV-6A and HHV-6B and HHV-7 viral DNA in CSF of AD patients are below the detection limit. It has been reported that the amount of HHV-6A and HHV-7 DNA in the brain of AD patients (parahippocampal gyrus, etc.) was detected twice as much as that of non-AD patients. Is desirable.
As a preemptive therapy for HHV-6 encephalitis with acute and severe symptoms, foscarnet is administered at the highest dose (180 mg/kg/day) for several days (minimum 3 days) and HHV-6B in cerebrospinal fluid. There is a report that the DNA of the above was able to be below the detection limit.
However, in the case of AD, although it is certainly common with HHV-6 encephalitis as a cognitive mechanism lowering symptom, unlike HHV-6/7 high-concentration infection/highly active HHV-6 encephalitis, it is considered to progress mildly. Has been. Therefore, the dosage regimen of foscarnet in treating AD is likely to be fairly modest.
The latent form of HHV-6 cannot completely suppress its reactivation. It is also possible to select a dosage and a method for preservative treatment, in which the amount of viral DNA in the healthy person or in the cerebrospinal fluid is below the detection limit, and then the patient is switched to maintenance therapy to improve patient compliance.
　本明細書において「有効量」とは、脳神経系の細胞におけるHHV-6/7の感染レベルを少なくとも上昇させないために必要な用量であり、感染レベルはCSF中のHHV-6/7由来のDNAの蓄積レベルとして評価できるものである。具体的な用量は、患者におけるウイルス感染の状況や症状、身体的条件、投与経路、抗ウイルス剤の各ウイルスに対する効力などによって異なる。
　ADに対するホスカルネット製剤の治療においては、早期に脳内のHHV-6A及びHHV-6B並びにHHV-7の感染レベルを健常人レベルあるいは検知水準以下とし、その水準を維持することが望ましい。
　上述のように、ホスカルネットのHHV-6脳炎に対する適応において、発症後のフルドーズ（180mg/kg/日）の点滴静注が優れた効果を示していることから、例えば、AD患者に対し、入院の上、有効成分としてホスカルネットナトリウム水和物を6g含有する250mLバイアル（24mg/mL）の注射剤（「ホスカビル（登録商標）注」製剤）を先制療法剤とする。
　先制療法の初回投与の有効量の最高用量は180mg/kg/日を基準とするが、腎障害を併発する患者やコンプライアンスの確保の為に髄腔内投与を行う場合、有効量は適宜補正される。維持療法としての入院期間中の有効量として、「ホスカビル（登録商標）注」の製剤を1000mLの生理食塩水に溶解し、1回125~250mL（ホスカルネットとして0.75～1.5g）を目安に投与する。また、状況に応じて静注あるいは点滴静注も考慮する。 In the present specification, the "effective amount" is a dose required to at least not increase the infection level of HHV-6/7 in cells of the cranial nervous system, and the infection level is HHV-6/7-derived DNA in CSF. It can be evaluated as the accumulation level of. The specific dose varies depending on the condition and symptoms of viral infection in the patient, physical condition, administration route, efficacy of antiviral agent against each virus, and the like.
In the treatment of AD with foscarnet preparation, it is desirable to maintain the level of HHV-6A, HHV-6B, and HHV-7 in the brain at an early stage, which is equal to or lower than the level of healthy individuals or the level of detection.
As described above, in the adaptation of foscarnet to HHV-6 encephalitis, the post-onset full dose (180 mg/kg/day) intravenous infusion shows an excellent effect, for example, for AD patients, After hospitalization, a 250 mL vial (24 mg/mL) injection (“Foscavir (registered trademark) injection” formulation) containing 6 g of foscarnet sodium hydrate as an active ingredient is used as a preemptive agent.
The maximum effective dose for the first dose of preemptive therapy is 180 mg/kg/day as a standard, but the effective dose may be adjusted appropriately if it is administered intrathecally in patients with renal impairment or to ensure compliance. It As an effective dose during maintenance as a maintenance therapy, the formulation of "Foscavir (registered trademark)" is dissolved in 1000 mL of physiological saline, and 125 to 250 mL (0.75 to 1.5 g as foscarnet) is used as a guideline. Administer. In addition, consider IV or IV infusion depending on the situation.
　また、例えばホスカルネットとして500mgないし1500mg含有の経口剤を調製し、退院後の維持療法に用いる。持続療法ではホスカルネットとして2000～6000mgを1日に3ないし4回に分けて投与する。その期間はADの症状（特に認知機能の障害）が消失するまでとするが、3ないし6ヵ月を目途とする。また、ホスカルネットは粘膜刺激性を有することから、その経口剤の投与に際しては、テプレノン、スクラルファート、アズレンスルフォル酸ナトリウム、レパミピド、ポラプレジンク、イルソグラジンマイレン酸塩、ベキサネート塩酸塩、ソファルコン、セトラキサート塩酸塩、エカベトナトリウム水和物等の胃粘膜保護作用のある薬剤との併用あるいはそれらとの合剤が望ましい。 Also, for example, prepare an oral preparation containing 500 mg to 1500 mg of foscarnet and use it for maintenance therapy after discharge. In continuous therapy, 2000 to 6000 mg of foscarnet is administered in 3 to 4 divided doses daily. The period is until the symptom of AD (especially cognitive impairment) disappears, but it is expected to be 3 to 6 months. In addition, since foscarnet has mucosal stimulant properties, teprenone, sucralfate, sodium azulensulforate, repamipide, polaprezinc, irsogladine maleate, bexanate hydrochloride, sofarcone, when administering its oral preparation, It is desirable to use in combination with a drug having a gastric mucosa-protecting action such as cetraxate hydrochloride and ecabet sodium hydrate, or a combination thereof.
　なお、定期的（1～3ヵ月に一度）に脳脊髄液中又は血漿中又は唾液中のHHV-6A、HHV-6B及びHHV-7のDNA量をモニターし、基準を超えている場合、再度入院の上、先制療法に準じたホスカルネットの投与を行う。投与量は患者の状態や検知されたウイルス量により、適宜調整する。 It should be noted that the DNA amount of HHV-6A, HHV-6B and HHV-7 in cerebrospinal fluid, plasma or saliva should be monitored regularly (once every 1 to 3 months), and if it exceeds the standard, it should be checked again. After being hospitalized, administer foscarnet according to pre-emptive therapy. The dose should be adjusted appropriately according to the condition of the patient and the amount of virus detected.
　ADは高齢者に発症し、高齢者では腎機能が低下している場合が多い。高用量での投与の場合は、利尿剤や投与後の十分な水の補給により腎障害に関するリスクを軽減することが望ましい。
　また、腎障害が懸念される場合は、髄腔内投与によって投与量を減らす方法の他、尿クレアチンの値に応じたホスカルネット投与量の変更が望ましい。この投与量の変更の方法はルーチン化しており、HHV-6脳炎に対する治療ガイドライン（参考文献３２）にその詳細が記載されている。ADに対するホスカルネット療法においても、このガイドラインが応用できる。 AD occurs in the elderly, and renal function is often reduced in the elderly. For high doses, it is desirable to reduce the risk of renal damage by diuretics and adequate water supplementation after administration.
Further, when renal damage is a concern, it is desirable to change the dose of foscarnet according to the value of urinary creatine, in addition to the method of reducing the dose by intrathecal administration. The method of changing the dose is routine and is described in detail in the treatment guideline for HHV-6 encephalitis (reference document 32). This guideline can also be applied to foscarnet therapy for AD.
　以下に実施例を示し、本発明を具体的に説明するが、本発明はこれらに限定されるものではない。
　本発明におけるその他の用語や概念は、当該分野において慣用的に使用される用語の意味に基づくものであり、本発明を実施するために使用する様々な技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易且つ確実に実施可能である。また、各種の分析などは、使用した分析機器又は試薬、キットの取り扱い説明書、カタログなどに記載の方法を準用して行った。
　なお、本明細書中に引用した技術文献、特許公報及び特許出願明細書中の記載内容は、本発明の記載内容として参照されるものとする。 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.
Other terms and concepts in the present invention are based on the meanings of terms commonly used in the art, and various technologies used for carrying out the present invention are notably defined as those demonstrating the source thereof. Except for this, those skilled in the art can easily and surely carry out the implementation based on publicly known documents and the like. In addition, various analyzes and the like were carried out by applying methods described in analytical instruments or reagents used, instruction manuals of kits, catalogs and the like.
The description in the technical documents, patent publications, and patent application specifications cited in this specification shall be referred to as the description of the present invention.
（参考例１－１） ホスカルネットのHHV-6に対する効果－１
　ヒトTリンパ球芽細胞株HSB-2（ATCC社）にHHV-6を接種、感染を成立させた後に、ホスカルネット三ナトリウム６水和物（以下、本参考例では「ホスカルネット」と言う。以下の参考例及び実施例も同様。）67μMを添加し14日培養した。生細胞数、及び蛍光抗体法によるHHV-6抗原陽性細胞を計数した。
　結果は以下に示すように、ホスカルネットはHHV-6による細胞障害及びウイルス抗原陽性細胞の実現を67μMで完全に抑制した。 (Reference Example 1-1) Effect of foscarnet on HHV-6-1
Human T lymphoblastoid cell line HSB-2 (ATCC) was inoculated with HHV-6 to establish infection, and then foscarnet trisodium hexahydrate (hereinafter referred to as "foscarnet" in this reference example). The same applies to the following Reference Examples and Examples.) 67 μM was added and the cells were cultured for 14 days. The number of viable cells and HHV-6 antigen-positive cells by the fluorescent antibody method were counted.
As shown in the results below, foscarnet completely suppressed HHV-6-induced cytotoxicity and virus antigen-positive cells at 67 μM.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
（参考例１－２）、ホスカルネットのHHV-6に対する効果－２
　ホスカルネット(Foscarnet)と汎用されている他のヘルペスウイルス剤（Acyclovir，Penciclovir、Ganciclovir）とのHHV-６に対する効果をヒト由来のT-lymphoblast cells 及びcord blood lymphocytesを用いた比較において、以下のような結果が報告されている（非特許文献１７）。 (Reference Example 1-2), Effect of foscarnet on HHV-6-2
The effects of foscarnet and other commonly used herpesviruses (Acyclovir, Penciclovir, Ganciclovir) on HHV-6 in human T-lymphoblast cells and cord blood lymphocytes were compared as follows. Such results have been reported (Non-Patent Document 17).
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
ホスカルネットは、他の抗ウイルス剤と比較して抗HHV-6 活性が極めて高く、かつ有効量と毒性量との乖離が最も大きく安全性に優れている。 Foscarnet has extremely high anti-HHV-6 activity as compared with other antiviral agents, and has the greatest difference between the effective dose and the toxic dose, and is excellent in safety.
（参考例２）ホスカルネットのHHV-6A及びHHV-6Bに対する他剤との比較
　HHV-6A及びHHV-6Bに対するホスカルネットの効果を他の抗ウイルス剤と比較すべく、ヒトTリンパ球芽細胞株HSB-2細胞（ATCC社）及びMolt-3細胞（ABI社）にそれぞれHHV-6A GS株（NIH）あるいはHHV-6B Z29株（ABI社）を感染させ、抗ウイルスアッセイと細胞毒性アッセイを行った。すなわち、抗ウイルスアッセイは、それぞれの細胞に10６細胞あたり100CCID50のHHV-6を感染させて10％FCS、2mM L-グルタミン、0.1％重炭酸ナトリウムを添加したRPMI培地で培養し、37℃で90分間吸収させた。洗浄、細胞数調製の後、調整した抗ウイルス剤それぞれ連続希釈を行って添加した。3～4日ごとに抗ウイルス剤を添加した培地に交換し、10～12日目にウイルス増殖が50％まで抑制された抗ウイルス剤の濃度を算出しCC50とした。（非特許文献１６） (Reference Example 2) Comparison of foscarnet with other agents against HHV-6A and HHV-6B Human T lymphocytes were compared in order to compare the effect of foscarnet against HHV-6A and HHV-6B with other antiviral agents. Blast cell line HSB-2 cells (ATCC) and Molt-3 cells (ABI) were infected with HHV-6A GS (NIH) or HHV-6B Z29 (ABI), respectively, and antiviral assay and cytotoxicity were performed. The assay was performed. That is, the antiviral assay was carried out by infecting each cell with 100 CCID 50 of HHV-6 per 10 6 cells, culturing the cells in RPMI medium supplemented with 10% FCS, 2 mM L-glutamine, and 0.1% sodium bicarbonate at 37°C. Absorbed for 90 minutes. After washing and cell number adjustment, the adjusted antiviral agents were serially diluted and added. The medium was replaced with an antiviral agent-added medium every 3 to 4 days, and the concentration of the antiviral agent in which the viral growth was suppressed to 50% was calculated on the 10th to 12th day and defined as CC 50 . (Non-patent document 16)
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
（参考例３）HHV-7に対するホスカルネットと他の抗ウイルス剤の効果
　　ホスカルネットのHHV-7に対する効果をヒト由来のSup-T1 cell line 及びpurified CD4+ T lymphocytesを用いて、他の抗ウイルス剤と比較した結果は以下の通りである（非特許文献１７）。 (Reference Example 3) Effect of foscarnet on HHV-7 and other antiviral agents The effect of foscarnet on HHV-7 was examined by using human-derived Sup-T1 cell line and purified CD4+ T lymphocytes. The results of comparison with the viral agent are as follows (Non-patent document 17).
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
以上に示すようにホスカルネットはHHV-6A及びHHV-7に対する約２倍の有効量でHHV-6Bに対する抗ウイルス作用を有し、標的ウイルスごとに投与量を大きく変える必要がなく、バランスのとれた抗ウイルス剤である（図３）。 As shown above, foscarnet has an antiviral effect against HHV-6B in an effective dose about twice that of HHV-6A and HHV-7, and it is not necessary to significantly change the dose for each target virus, and to maintain the balance. It is a taken antiviral agent (Fig. 3).
（参考例４）ホスカルネットの抗マルチウイルス活性
　ホスカルネットは以下のような各種ウイルスに対する抗ウイルス効果がある（非特許文献１７）。特にCMV、HSV-1、EBV、HIV-1などは脳内での潜伏感染が知られており、活性化した場合、脳炎を惹起することが知られている。従って、AD治療薬としての抗ウイルス剤には、かかるウイルスに対する効果を有することが望ましい（クリニジェン株式会社、「ホスカビル（登録商標）・インタビューフォーム2019年3月版」、2019）。 (Reference Example 4) Anti-multivirus activity of foscarnet Foscarnet has an antiviral effect against the following various viruses (Non-patent document 17). In particular, CMV, HSV-1, EBV, HIV-1 and the like are known to have latent infection in the brain, and when activated, they are known to cause encephalitis. Therefore, it is desirable that an antiviral agent as a therapeutic agent for AD has an effect against such a virus (Clinigen Co., Ltd., “Foscavir (registered trademark) Interview Form March 2019 edition”, 2019).
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
（参考例５）　ホスカルネットの脳への移行性
ホスカルネットの脳脊髄液への移行
　AIDS患者（n=27）にホスカルネットを56～213mg/kg（中間値：100mg/kg）を2～6時間静脈内注入したとき、脳脊髄液中のホスカルネットの濃度は、注入直後に50～250nmol/mLを示し、血漿中濃度の10～70%に相当した。投与6時間後以降における脳脊髄液中濃度は、血中濃度とほぼ同等であった。（非特許文献１５、図４） (Reference Example 5) Transfer of foscarnet to the brain Transfer of foscarnet to cerebrospinal fluid 56 to 213 mg/kg (intermediate value: 100 mg/kg) of foscarnet to AIDS patients (n=27) When injected intravenously for 2 to 6 hours, the concentration of foscarnet in cerebrospinal fluid was 50 to 250 nmol/mL immediately after the injection, corresponding to 10 to 70% of the plasma concentration. The cerebrospinal fluid concentration 6 hours after administration was almost the same as the blood concentration. (Non-patent document 15, FIG. 4)
（参考例６）　ホスカルネットのカルシウムに対する作用
（６－１）ホスカルネットの遊離カルシウムに対する作用－神経節接合部に及ぼす影響
　ラット摘出横隔膜神経節標本を用いて、ホスカルネットの電気刺激誘発収縮に対する影響を検討した。また、ホスカルネット添加した時の栄養液中の遊離カルシウムの濃度を測定した。その結果、ホスカルネットは0.1mM から3mM まで影響を及ぼさなかった。10mMでは10分後に平均14％の収縮の抑制が観察されたが、統計的有意差はなかった。実験終了時に採取したホスカルネット10mMを含む栄養液中のCa＋＋イオン濃度はコントロールに比較して7％減少していた。
　以上、ラット摘出横隔膜神経節標本において、ホスカルネットは10mMで電気刺激誘発収縮の抑制傾向を示した。 Reference Example 6 Effect of Foscarnet on Calcium (6-1) Effect of Foscarnet on Free Calcium-Effect on Ganglion Junction Induction of Electrical Stimulation of Foscarnet Using Isolated Rat Diaphragm Ganglion Specimens The effect on contraction was investigated. In addition, the concentration of free calcium in the nutrient solution when foscarnet was added was measured. As a result, foscarnet had no effect from 0.1 mM to 3 mM. An average of 14% inhibition of contraction was observed after 10 minutes at 10 mM, but there was no statistically significant difference. The Ca ++ ion concentration in the nutrient solution containing 10 mM foscarnet collected at the end of the experiment was reduced by 7% compared to the control.
As described above, foscarnet showed a tendency to suppress electrical stimulation-induced contraction at 10 mM in rat isolated diaphragm ganglion specimens.
（６－２）ホスカルネットのカルシウム恒常性に与える影響
　イヌにホスカルネット405mg/kgを静脈内投与したとき、総血清カルシウム値が2.5mmol/Lから2.0mml/Lに、遊離カルシウムの濃度が1.3mmol/Lから0.9mmol/Lに低下した。この時、1／3例で、振戦を伴った頭部及び頸部の筋トーヌスが実現した。810mg/kgの投与では総血清カルシウム値が2.5mmol/L から 1.6mmol/L に低下し、遊離カルシウム濃度が 1.3 mmol/L から 0.7mmol/L に低下し、筋攣縮が観察された。
　尚、遊離カルシウム濃度の低下及び筋攣縮等の症状は、投与終了後24時間にはすべて消失した。
　ヒトにおいても、CMV網膜炎患者にホスカルネットを90及び120mg/kgを静脈内単回投与した試験で、用量依存的に血清カルシウム値の低下が認められた（図５）。この時、120mg/kgを投与した11例中2例で低カルシウム血症によると思われる口周囲の刺激痛四肢のしびれ、麻痺等が観察された（クリニジェン株式会社、「ホスカビル（登録商標）・インタビューフォーム2019年3月版」、2019）。  (6-2) Effect of foscarnet on calcium homeostasis When 405 mg/kg of foscarnet was intravenously administered to dogs, the total serum calcium level was changed from 2.5 mmol/L to 2.0 mml/L, and the concentration of free calcium was increased. Decreased from 1.3 mmol/L to 0.9 mmol/L. At this time, in 1/3 of the cases, muscle tonus of the head and neck accompanied by tremor was realized. At 810 mg/kg, the total serum calcium level 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 spasm was observed.
Incidentally, symptoms such as a decrease in free calcium concentration and muscle spasms disappeared 24 hours after the end of administration.
In humans as well, in a test in which CMV retinitis patients were administered with foscarnet 90 and 120 mg/kg once intravenously, a decrease in serum calcium level was observed in a dose-dependent manner (FIG. 5). At this time, irritation pain around the mouth, numbness of the extremities, paralysis, etc., which is considered to be due to hypocalcemia, was observed in 2 out of 11 patients administered 120 mg/kg (Clinigen Co., Ltd., “Foscavir (registered trademark). Interview form March 2019 edition, 2019).
(参考例７)　ホスカルネットの経口吸収性
（７－１）実験動物での経口吸収性
　マウス（24mg/kg）、ラット（24、96mg/kg）、イヌ（60mg/kg）に14C－ホスカルネットを滅菌水に溶かして経口投与した結果、吸収率はいずれも約50％で、Tmaxは15分(マウス)、30分(ラット)、1.4時間(イヌ)で、T1/2は約8時間（マウス、ラット）、33時間（イヌ）であった。(クリニジェン株式会社、「ホスカビル（登録商標）・インタビューフォーム2019年3月版」、2019） Reference Example 7 Oral Absorption of Foscarnet (7-1) Oral Absorption in Experimental Animals 14 C-in mice (24 mg/kg), rats (24, 96 mg/kg) and dogs (60 mg/kg) As a result of oral administration of foscarnet dissolved in sterile water, the absorption rate was about 50% in all cases, T max was 15 minutes (mouse), 30 minutes (rat), 1.4 hours (dog), T 1/2 Was about 8 hours (mouse, rat) and 33 hours (dog). (Clinigen Co., Ltd., "Foscavir (registered trademark) Interview Form March 2019 Edition", 2019)
（７－２）ヒトでの経口吸収性
　CMV網膜炎を併発したAIDS患者6例に4gのホスカルネットを6時間間隔で3日間経口投与した時の吸収率（尿中排泄より測定）は下記（表９：Source : Table IV, 非特許文献１５）に示されるように約18％であった。 (7-2) Oral absorbability in humans The absorption rate (measured from urinary excretion) after oral administration of 4 g of foscarnet for 6 days to 6 AIDS patients with CMV retinitis was as follows. It was about 18% as shown in (Table 9: Source: Table IV, Non-Patent Document 15).
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
（実施例１）　ホスカルネットによる、ウイルス（HHV-6,HHV-7）感染ヒト由来神経細胞でのアミロイドβ（オリゴマーを含む）の細胞内取り込み抑制作用
　本試験では、アミロイドβ（Aβ）のウイルス（HHV-6,HHV-7）感染細胞内への取り込み、及びホスカルネットのよる細胞内への取り込み抑制効果を調べた。本試験の手順は以下に従った。
（１－１）HHV-6A,HHV-6B及びHHV-7各ウイルス溶液の調製
　以下の試験材料及び手順に従って、試験の用いる上記各ウイルス溶液を作製した
（ａ）試験材料
１）試験に用いた以下の細胞株はHHV-6 Foundation （米国・カリフォルニア州サンタ・バーバラ市）から提供を受けた。
・HSB-2株：HSB-2 Uninfected Cell, Master Seed, 1.0 mL, @1×107 cells, 12/17/2018, (BIOCELL Diagnostics H10-865)； 10% FBS,1×ATB添加のIMDM培地を用いて培養（参考：CCRF-HSB-2(ATCC CCL-120.1) Product Sheet）
・Sup-T1株:  Sup-T1 Uninfected Cell, Master Seed, 1.0 mL, @1×107 cells, 12/17/2018, (BIOCELL Diagnostics H10-864)； 10% FBS,1×ATB添加のRPMI-1640培地を用いて培養（参考：Sup-T1(ATCC CRL-1942) Product Sheet）
・HHV-6A/HSB-2株： Master Seed, HHV-6A, GS strain, infected HSB-2 cells,1.0 mL, @5×106 cells/mL, 8/13/2018,　(BIOCELL Diagnostics H10-849)；HSB-2と同様の培地（10% FBS,1×ATB添加のIMDM培地）で培養
・HHV-6B/Sup-T1 株 : Sup-T1 Master seed, HHV-6B, Z29 strain, infected Sup-T1 cells, 1.0 mL, @5×106 cells/mL, 8/16/2018 (BIOCELL Diagnostics H10-850)； Sup-T1と同様の培地（10% FBS,1×ATB添加のRPMI-1640培地）で培養
・HHV-7/Sup-T1株 ： Master seed, HHV-7, JI strain, P1,infected Sup-T1 cells, 1.0 mL, @5×10６ cells/mL, Early passage, 12/2/2018 (BIOCELL Diagnostics H10-859)； Sup-T1と同様の培地（10% FBS,1×ATB添加のRPMI-1640培地）で培養。
２）FBS： Fetal Bovine Serum,qualified, Brazil(lot 42Q6170K:Thermo Fisher Scientific 
10270)
３）ABT :  抗生物質・抗真菌剤混合溶液（lot L7P3293：Nacalai tesque 09366-44）
４）IMDA培地 :　IMDA, GlutaMAX Supplement　（lot 2003869： Thermo Fisher Scientific 31980-030）
５）RPMI-1640培地：　RPMI-1640 Medium, GlutaMAX Supplement（lot 1967676：Thermo Fisher Scientific 61870-036） (Example 1) Inhibitory effect of foscarnet on intracellular uptake of amyloid β (including oligomer) in virus-(HHV-6, HHV-7)-infected human-derived neurons In this test, amyloid β (Aβ) The effect of uptake into cells infected with viruses (HHV-6, HHV-7) and uptake into cells by foscarnet was investigated. The procedure of this test was as follows.
(1-1) Preparation of HHV-6A, HHV-6B and HHV-7 Virus Solutions Each virus solution used in the test was prepared according to the following test materials and procedures (a) Test material 1) Used for the test The following cell lines were provided by the HHV-6 Foundation (Santa Barbara, California, USA).
・HSB-2 strain: HSB-2 Uninfected Cell, Master Seed, 1.0 mL, @1×10 7 cells, 12/17/2018, (BIOCELL Diagnostics H10-865); 10% FBS, 1×ATB added IMDM medium (Reference: CCRF-HSB-2(ATCC CCL-120.1) Product Sheet)
・Sup-T1 strain: Sup-T1 Uninfected Cell, Master Seed, 1.0 mL, @1×10 7 cells, 12/17/2018, (BIOCELL Diagnostics H10-864); RPMI- supplemented with 10% FBS, 1×ATB Culture using 1640 medium (reference: Sup-T1(ATCC CRL-1942) Product Sheet)
・HHV-6A/HSB-2 strain: Master Seed, HHV-6A, GS strain, infected HSB-2 cells, 1.0 mL, @5×10 6 cells/mL, 8/13/2018, (BIOCELL Diagnostics H10-849 ); Culture in the same medium as HSB-2 (IMDM medium containing 10% FBS, 1xATB) HHV-6B/Sup-T1 strain: Sup-T1 Master seed, HHV-6B, Z29 strain, infected Sup- T1 cells, 1.0 mL, @5×10 6 cells/mL, 8/16/2018 (BIOCELL Diagnostics H10-850); Medium similar to Sup-T1 (10% FBS, RPMI-1640 medium with 1×ATB added) Cultivated in HHV-7/Sup-T1 strain: Master seed, HHV-7, JI strain, P1,infected Sup-T1 cells, 1.0 mL, @5×10 6 cells/mL, Early passage, 12/2/2018 (BIOCELL Diagnostics H10-859); Cultured in the same medium as Sup-T1 (RPMI-1640 medium supplemented with 10% FBS, 1×ATB).
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）非感染細胞の培養
　ウイルス溶液を取得すべく、HHV-Aに関しては非感染のHSB-2株、HHV-6B及びHHV-7に関してはSup-T1株を、以下の手順で培養。
１） 凍結保存細胞のチューブを37℃で温浴にて急速解凍し、HSB-2細胞については10% FBS, 1×ATB添加のIMDM培地で、Sup-T1細胞については10% FBS, @1×ATB添加のRPMI-1640培地（各9mL）と混合。
２）上記各混合液を遠心エバポレーター（S/N 11600775； EYELA CVE-2200）で遠心分離（300×g、5 min）し、上清を廃棄する。
３）上清廃棄後の細胞ペレットをタッピングでほぐした後、それぞれ新しい培地で懸濁。
４）HSB-2、Sup-T1を、@2~3×105  cells/mLの細胞密度で浮遊培養容器に播種し、37℃、5％CO2で培養。@1~2×106 cells/Lになるまで増殖したら、それぞれについて新しい培地で3～5倍に希釈して継代。 (B) Culturing of non-infected cells In order to obtain a virus solution, non-infected HSB-2 strain for HHV-A and Sup-T1 strain for HHV-6B and HHV-7 were cultured by the following procedure.
1) A tube of cryopreserved cells was rapidly thawed in a warm bath at 37°C, 10% FBS for HSB-2 cells and IMDM medium supplemented with 1xATB, and 10% FBS, @1x for Sup-T1 cells. Mix with RPMI-1640 medium (9 mL each) supplemented with ATB.
2) Centrifuge (300 xg, 5 min) the above mixture with a centrifugal evaporator (S/N 11600775; EYELA CVE-2200) and discard the supernatant.
3) After loosening the supernatant, loosen the cell pellet by tapping and suspend each with a new medium.
4) HSB-2 and Sup-T1 were seeded in a suspension culture container at a cell density of @2 to 3 × 10 5 cells/mL, and cultured at 37°C and 5% CO 2 . After growing to @ 1 to 2 × 10 6 cells/L, dilute them 3 to 5 times with fresh medium and pass.
（ｃ）ウイルス感染細胞の培養
　米国のHHV-6 Foundation が設定した「Protocol for high titer virus propagation, http://hhv6fiundatio.org(RESEARCH-Lab).Protocols: 2019.02.08)に従って、各試験ウイルスに感染した細胞を培養。
１） HHV-6A/HSB-2（HHV-6Aに感染したHSB-2株）並びにHHV-6B/Sup-T1（HHV-6Bに感染したSup-T1株）及びHHV-7/Sup-T1（HHV-7に感染したSup-T1株）について、凍結保存細胞（各5×106 cells ）を37℃で温浴にて急速解凍。
２）等量（5×106 cells）の非感染細胞（HHV-6A/HSB-2に対してはHSB-2、HHV-6B/Sup-T1及びHHV-7/Sup-T1に対してはSup-T1、以下同様）と混合して全量5mLとし、T-75 フラスコ2本に分注（2.5mL/フラスコ）。
３）各フラスコを傾けた状態で穏やかに撹拌しながら2時間培養（37℃、5％ CO2）。
４）各フラスコ10 mLの新しい培地を添加。
５）37℃、5% CO2で培養。 (C) Culturing of virus-infected cells Each test virus was tested according to "Protocol for high titer virus propagation, http://hhv6fiundatio.org(RESEARCH-Lab).Protocols: 2019.02.08" set by HHV-6 Foundation in the United States. Culture infected cells.
1) 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 ( Cryopreserved cells (5×10 6 cells each) of Sup-T1 strain infected with HHV-7 were rapidly thawed in a warm bath at 37°C.
2) Equal amount (5×10 6 cells) of non-infected cells (HSB-2 for HHV-6A/HSB-2, HHV-6B/Sup-T1 and HHV-7/Sup-T1) Sup-T1, the same as below) to a total volume of 5 mL, and dispensed into 2 T-75 flasks (2.5 mL/flask).
3) Incubate for 2 hours with each flask tilted and gently agitated (37°C, 5% CO 2 ).
4) Add 10 mL of fresh medium to each flask.
5) Incubate at 37°C, 5% CO 2 .
（ｄ）ウイルス感染率の算定
　感染細胞のウイルス感染率については、定期的にTC20全自動セルカウンター（S/N 508BR1149; Bio-Rad）にてトリパンブルー染色した細胞の数を測定（全細胞数、生細胞数；細胞サイズ範囲：7～36μm、12～36μm[>12μm]）。非感染細胞（HSB-2またはSup-T1）の測定結果と比較し、次の式を用いて感染率を算出。
感染生細胞数＝感染細胞の生細胞数×（感染細胞の>12μm生細胞数－非感染細胞の>12μm生細胞数の割合）
感染細胞特異的な死細胞数＝感染細胞の全細胞数×（非感染細胞の生存率－感染細胞の生存率）
感染率（％）＝（感染細胞数＋感染細胞特異的な死細胞数）／感染細胞の全細胞数×100 (D) Calculation of virus infection rate Regarding the virus infection rate of infected cells, the number of trypan blue stained cells was periodically measured with a TC20 fully automatic cell counter (S/N 508BR1149; Bio-Rad) (total cell number). , Viable cell number; cell size range: 7-36 μm, 12-36 μm [>12 μm]). Calculate the infection rate using the following formula by comparing with the measurement results of uninfected cells (HSB-2 or Sup-T1).
Infected live cell count = Infected cell viable cell count x (ratio of infected cell >12 μm viable cell-non-infected cell >12 μm viable cell)
Number of dead cells specific to infected cells = total number of infected cells x (survival rate of uninfected cells-survival rate of infected cells)
Infection rate (%) = (number of infected cells + number of dead cells specific to infected cells) / total number of infected cells x 100
（ｅ）ウイルス溶液の調製
（ｅ－１）手順
　HSB-2又はSup-T1（等量～5倍程度）を適量の新しい培地とともに添加して継代し、感染率がおよそ60～80％になるまで培養を継続。その後、細胞を培地とともに50 mL遠心管に回収し、凍結保存（－80℃）。その後、以下の手順でウイルス溶液の調製を行う。
１） 37℃で温浴して急速解凍し、ボルテックスミキサーで30秒間混合。凍結保存後、急速解凍し、ボルテックスミキサーで混合を同様に更に一回。
２）遠心分離（1,500 rpm、10 min、4℃）、上清（SN1）と沈殿（PT1）を回収。
３）SN1を同様に遠心分離し、上清（SN2）と沈殿（PT2）を回収。
４）PT1、PT2をそれぞれ1mLの培地に懸濁し、あわせて混合（PT3）。
５）PT3をドライアイス・エタノールで急速凍結後、37℃で温浴にて急速解凍し、これを計2回行う。
６）PT3を遠心分離し（1,500 rpm、10 min、4℃）、上清を回収（SN4）。
７）SN2とSN3を混合して遠心分離（4,500 rpm、20 min、4℃）し、上清を回収する（これをSN4とする）。
８）SN4を0.45μmをフィルターにろ過後、遠心型限外ろ過フィルターにて濃縮。200～300μLずつ分注して凍結保管（－80℃：ウイルス溶液） (E) Preparation of virus solution (e-1) Procedure HSB-2 or Sup-T1 (equivalent to about 5 times) is added together with an appropriate amount of fresh medium and subcultured to increase the infection rate to about 60 to 80%. Continue culturing until. Then, collect the cells together with the medium in a 50 mL centrifuge tube and store them frozen (-80°C). Then, a virus solution is prepared by the following procedure.
1) Heat thaw at 37℃ for rapid thawing and mix for 30 seconds with a vortex mixer. After cryopreservation, quickly thaw and mix once more with a vortex mixer.
2) Centrifuge (1,500 rpm, 10 min, 4°C) and collect the supernatant (SN1) and precipitate (PT1).
3) Centrifuge SN1 in the same manner and collect the supernatant (SN2) and precipitate (PT2).
4) Suspend PT1 and PT2 in 1mL of medium and mix them together (PT3).
5) PT3 is rapidly frozen with dry ice/ethanol and then rapidly thawed at 37°C in a warm bath, and this is performed twice in total.
6) Centrifuge PT3 (1,500 rpm, 10 min, 4°C) and collect the supernatant (SN4).
7) SN2 and SN3 are mixed and centrifuged (4,500 rpm, 20 min, 4°C), and the supernatant is collected (this is designated as SN4).
8) After filtering 0.45 μm of SN4 through a filter, concentrate it with a centrifugal ultrafiltration filter. Aliquot 200 to 300 μL and store frozen (-80℃: virus solution)
（ｅ－２）ウイルス溶液中のウイルスGCの測定
　前記により得られた各ウイルス溶液50μLについて、ウイルスの遺伝子数（GC）について以下の手順で測定した。
１）DNeasy Blood & Tissue Kit (lot 151046692: QIAGEN 69504) のPurification of Total DNA from Animal Blood or Cells (Spin-Column Protocol)の手順に従って、RNase A処理を含めたカラム添加直前までのサンプル調整を行い、Monarch DNA Cleanup Columnsを用いてMonarch PCR & DNA Cleanup Kit（lot 0071711: NEB T1030L）の手順に従ってDNAを調整。
２）1μLの各ウイルス溶液より抽出したDNAを鋳型とし、0.3μMプライマー（以下の表に示す）、１×SsoAdvanced Universal SYBR Green Supermix (lot 64098857: Bio‐Rad 1725271)の総量8μLの反応条件でReal-time PCR 解析を実施。CFX Connect Real-Time PCR Detection System(S/N 788BR04674: Bio-Rad) を測定機器として使用。 (E-2) Measurement of virus GC in virus solution With respect to 50 μL of each virus solution obtained above, the number of virus genes (GC) was measured by the following procedure.
1) According to the procedure of Purification of Total DNA from Animal Blood or Cells (Spin-Column Protocol) of DNeasy Blood & Tissue Kit (lot 151046692: QIAGEN 69504), perform sample preparation until column addition including RNase A treatment, Prepare the DNA using Monarch DNA Cleanup Columns according to the procedure of Monarch PCR & DNA Cleanup Kit (lot 0071711: NEB T1030L).
2) Using 1 μL of DNA extracted from each virus solution as a template, 0.3 μM primer (shown in the table below), 1×SsoAdvanced Universal SYBR Green Supermix (lot 64098857: Bio-Rad 1725271) under the reaction conditions of 8 μL in total. -Time PCR analysis is performed. CFX Connect Real-Time PCR Detection System (S/N 788BR04674: Bio-Rad) is used as a measuring instrument.
Figure JPOXMLDOC01-appb-T000018
 ３）濃度既知のHHV-6PCR産物（HHV-6Bを鋳型とし、HHV6-orf67-F1 プライマーとHHV6-orf67-R1プライマーでPCR増幅した産物をカラム精製したもの）または、HHV-7PCR産物（HHV-7を鋳型とし、Hs-ACTB-gF1プライマーと Hs-ACTB-gR1プライマーでPCR増幅した産物をカラム精製したもの）の希釈系列も並行して、Real -time PCR解析を行い、検量線（C1価と鋳型DNA数（ウイルスゲノムコピー数：GC）の関係を表す）を作成。その検量線に基づき、各ウイルス溶液中のGCを算出。その結果は以下の（表１１）の通りである。
Figure JPOXMLDOC01-appb-T000018
 3) HHV-6 PCR product with known concentration (HHV-6B as a template, PCR purified with HHV6-orf67-F1 primer and HHV6-orf67-R1 primer by column purification) or HHV-7 PCR product (HHV- 7 as a template, Hs-ACTB-GF1 primer and Hs-ACTB-gR1 that the product was PCR amplified with primers were column purified) dilution series is also in parallel to perform the Real -time PCR analysis, calibration curve (C 1 Create the relationship between the value and the number of template DNA (virus genome copy number: GC). Calculate the GC in each virus solution based on the calibration curve. The results are shown in (Table 11) below.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
（ｅ－３）力価の測定
　感染細胞でのウイルスの力価の測定は以下により行った。
１）未感染のHSB-2（HHV-6A対象）またはSup-T1（HHV-6B及びHHV-7対象）を3.3×105 cells/mLに調製し、0.45mL/well で24 well plate へ播種。一晩培養。
２）各ウイルス溶液50μLを培地で希釈して２0 ～  2-5 で希釈溶液を調製。
３）段階希釈したウイルス溶液50μLを未感染のHSB-2またはSup-T1に添加し（最終細胞密度は３×105 cells/mL）、2日間培養。
４）前記の「Aβ42の添加とウイルス感染」に定める手順により算定した感染率を算定してウイルス力価（IFU/mL）を決定した。 (E-3) Measurement of titer The titer of virus in infected cells was measured as follows.
1) Prepare uninfected HSB-2 (for HHV-6A) or Sup-T1 (for HHV-6B and HHV-7) at 3.3×10 5 cells/mL and seed 0.45 mL/well on a 24-well plate. .. Overnight culture.
2) preparing a diluted solution with 2 0-2 -5 was diluted with medium each virus solution 50 [mu] L.
3) Add 50 μL of serially 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) The virus titer (IFU/mL) was determined by calculating the infection rate calculated by the procedure specified in the above “Addition of Aβ 42 and virus infection”.
（ｅ－４）ウイルス溶液のウイルスゲノムコピー数（GC）の測定
　上記手順により得られた各ウイルス溶液中のウイルスゲノムコピー数（GC）は、以下の手順に従って測定した。
１）ウイルス溶液50μLよりゲノムDNAを調整。DNeasy Blood & Tissue Kit [Purification of Total DNA from Animal Blood or Cells(Spin-Column Protocol)]の手順に従って、RNase A 処理を含めたカラム添加直前までのサンプル調製を行う。
２）Monarch DNA Cleanup Columnを用いてMonarch PCR & DNA Cleanup Kit (lot 0071711;NEB T1030L)の手順に従ってDNAを精製。
３）１μLのウイルス溶液より抽出したDNAを鋳型とし、0.3μMプライマー、1×SsoAdvanced Universal SYBR Green Supermix の総量8μLの反応条件でReal-time PCR解析を実施。
４）CFX Connect Real-Time PCR Detection System(S/N 788BR04674; Bio-Rad)を測定機器として使用。 (E-4) Measurement of virus genome copy number (GC) of virus solution The virus genome copy number (GC) of each virus solution obtained by the above procedure was measured according to the following procedure.
1) Prepare genomic DNA from 50 μL of virus solution. According to the procedure of DNeasy Blood & Tissue Kit [Purification of Total DNA from Animal Blood or Cells (Spin-Column Protocol)], prepare the sample including RNase A treatment until just before column addition.
2) Purify DNA using the Monarch DNA Cleanup Column according to the procedure of Monarch PCR & DNA Cleanup Kit (lot 0071711; NEB T1030L).
3) Real-time PCR analysis was performed using 1 μL of the DNA extracted from the virus solution as a template under the reaction conditions of 0.3 μM primer and 1×SsoAdvanced Universal SYBR Green Supermix in a total amount of 8 μL.
4) CFX Connect Real-Time PCR Detection System (S/N 788BR04674; Bio-Rad) is used as a measuring instrument.
（１－２）Aβ42の調製
　細胞外のAβが細胞内に取り込まれるか否か、またそれにHHV-6A、HHV-6B及びHHV-7ウイルス感染が関与するか否かを試験する為、以下の手順によって試験用のAβ42を調整した。
１）Aβ42パウダー（Amyloid β- Protein (Human,1-42), 0.59mg;MW 4514.0; lot 680405, ペプチド研究所、4349-v)を室温に30分間静置。
２）ドラフトチャンバー内にて注射針を接続した注射筒にてHFIP（1,1,1,3,3,3Hexafluoro 
- 2 propanol; lot EKWML-ON; TCI H02424）を約131μL計り取り、Aβ42パウダー容器
のゴム栓を突き通して添加後、混合（1mM溶液）。ゴム 栓に注射針を突き刺して容器内部
の真空状態を開放。
３）室温で0.5～2時間静置（モノマー化）。
４）モノマー化混合液をタンパク質低吸着マイクロチューブ（1.5mL; Watson PK-15C-500）
2本に分注（66μL/tube）、遠心エバポレーター(S/N 11600775; EYELA CVE-2200)にて乾燥。冷凍保存（乾燥ペプチド：－80℃）。
５）上記乾燥ペプチドを室温に戻し、DMSO（Dimethyl sulfoxide: lot LKF233. Fujifilm Wako 037-24053）を13.1μL添加後、長音波破砕装置（Bioruptor UCD-250、S/N 250581, 
ソニック・バイオ株式会社）にて、10分間の160W,10 sec On/10 sec Off/30 cyclesで超音波処理により細胞を破砕し溶解。
６）上記容器液2μLずつを５本のマイクロチューブに分注して冷凍保存（5nM溶液：－80℃）
７）5mM溶液2mLをPBS(－)又は培地98μLと混合して100μM溶液を調整後、30秒間ボルテックスにて混合し、4℃で12～24時間静置（オリゴマー化）。 (1-2) Preparation of Aβ 42 In order to test whether extracellular Aβ is taken up into cells and whether HHV-6A, HHV-6B and HHV-7 viral infections are involved in it, Aβ 42 for the test was prepared by the procedure described in 1.
1) Aβ 42 powder (Amyloid β-Protein (Human, 1-42), 0.59 mg; MW 4514.0; lot 680405, Peptide Institute, 4349-v) was allowed to stand at room temperature for 30 minutes.
2) HFIP (1,1,1,3,3,3 Hexafluoro) with a syringe connected to an injection needle in the draft chamber.
-2 propanol; lot EKWML-ON; TCI H02424) was weighed out in an amount of about 131 μL, added through the rubber stopper of the Aβ 42 powder container, and then mixed (1 mM solution). Insert the injection needle into the rubber stopper to release the vacuum inside the container.
3) Let stand at room temperature for 0.5 to 2 hours (monomerization).
4) Low protein adsorption microtube (1.5mL; Watson PK-15C-500) of the monomer mixture.
Dispense into 2 tubes (66 μL/tube) and dry with a centrifugal evaporator (S/N 11600775; EYELA CVE-2200). Store frozen (dry peptide: -80℃).
5) After returning the dried peptide to room temperature and adding 13.1 μL of DMSO (Dimethyl sulfoxide: lot LKF233. Fujifilm Wako 037-24053), a long-wave sonication device (Bioruptor UCD-250, S/N 250581,
Sonic Bio Co., Ltd.) disrupts and lyses cells by sonication at 160 W for 10 minutes, 10 sec On/10 sec Off/30 cycles.
6) Dispense 2 μL each of the above container solution into 5 microtubes and store frozen (5 nM solution: -80°C)
7) 2 mL of 5 mM solution was mixed with PBS(-) or 98 μL of medium to prepare a 100 μM solution, which was mixed by vortex for 30 seconds and allowed to stand at 4° C. for 12 to 24 hours (oligomerization).
（１－３）試験サンプルの設定
　試験に用いるサンプルとして、以下を設定した。Aβ42は0.1～1,000nM(公比10の2段階希釈)と無添加の6条件、ホスカビル（登録商標）は終濃度800μMと無添加の2条件、ウイルス溶液はHHV-7のみ、HHV-7とHHV-6Aの混合、HHV-7とHHV-6Bの混合、無添加、それぞれMOI 0.1、1.0の8条件とし、以下の（表１２）の通りサンプル処理群を設定した。 (1-3) Setting of test sample The following was set as a sample used for the test. Aβ 42 is 0.1 to 1,000 nM (two-step dilution with a common ratio of 10) and 6 conditions without addition, foscavir (registered trademark) is 800 μM final concentration and 2 conditions without addition, virus solution is HHV-7 only, HHV-7 And HHV-6A were mixed, HHV-7 and HHV-6B were mixed, and no addition was carried out under MOI 0.1 and 1.0 conditions, respectively, and a sample treatment group was set as shown in (Table 12) below.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
（１－４）被験細胞の調製
　SH-SY5Y（lot:17C025,P11,19 April 2017; EACC 94030304）は0.1～10４cells/cm2の細胞密度で播種し、37℃、5％ CO２で培養。コンフルエントになるまで増殖したら、TrypLE Express (lot 1869186;thermo fisher Scientific 12604021)にて細胞を博させて懸濁し、継代。1.28×104cells/50μL/well(4×10４cells/cm2)を96-well plate 播種、10％FBS、1×NEAA、1×ATB添加「Ham’s F12」培地で1晩、培養。 (1-4) Preparation of test cells SH-SY5Y (lot:17C025,P11,19 April 2017; EACC 94030304) was seeded at a cell density of 0.1-10 4 cells/cm 2 , and at 37°C, 5% CO 2 . culture. After growing to confluence, trypLE Express (lot 1869186; thermo fisher Scientific 12604021) was used to expose and suspend the cells, and then passaged. 1.28×10 4 cells/50 μL/well (4×10 4 cells/cm 2 ) was seeded on a 96-well plate and cultured overnight in “Ham's F12” medium supplemented with 10% FBS, 1×NEAA and 1×ATB.
（１－５）Aβ42の添加とウイルス感染
　以下の手順により、被験細胞の培地にAβ42の添加及び被験細胞へのウイルス感染を施
行した。
１）「ホスカビル（登録商標）」（FCN, 点滴静注用ホスカビル（登録商標）注24mg/mL, 製造番号XX26、有効成分としてホスカルネット6gを含む）800μMの混合溶液25μLを調製。
２）前記により調製したウイルス溶液25μLを準備。
３）各被験サンプルを10％FBS、1×NEAA、1×ATB添加Ham’s F12培地（Ham’s F-12 with L-Glutamine and Phenol Red, lot TWH7034, Fujifilm Wako 087-08335）で24時及び48時間培養。
４）24時間後、被験サンプルの1／2について、波長450nmの吸光度を測定（プランク測定）。10μLのCCK-8（Cell Counting Kit-8, lot NT161, Dojindo 343-07623）を添加、1～4時間後に450nmの吸光度を測定。測定値からプランク測定値を減算した値（プランク減算値）を細胞数として評価。
５）48時間後、残りの１／２の被験サンプルについて、４）の手順で細胞数を測定。
６）吸光度測定後の細胞をPBS(－)で洗浄し、凍結保存（－80℃）。MOI 1.0、48時間の条件のみAβ42測定を実施。 (1-5) Addition of Aβ 42 and virus infection According to the following procedure, Aβ 42 was added to the medium of the test cells and the virus was infected to the test cells.
1) Prepare 25 μL of a mixed solution of 800 μM “foscavir (registered trademark)” (FCN, foscavir (registered trademark) injection 24 mg/mL for intravenous infusion, serial number XX26, containing 6 g of foscarnet as an active ingredient).
2) Prepare 25 μL of the virus solution prepared above.
3) Each test sample was cultured in 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, measure the absorbance at a wavelength of 450 nm for one-half of the test sample (Planck measurement). Add 10 μL of CCK-8 (Cell Counting Kit-8, lot NT161, Dojindo 343-07623), and measure the absorbance at 450 nm 1 to 4 hours later. The value obtained by subtracting the Planck measurement value from the measurement value (Planck subtraction value) is evaluated as the cell number.
5) After 48 hours, measure the number of cells in the remaining 1/2 test sample by the procedure of 4).
6) Wash the cells after measuring the absorbance with PBS(-) and store them frozen (-80°C). Aβ 42 measurement was performed only under conditions of MOI 1.0 and 48 hours.
（１－６）Aβ42の測定
　各被験サンプル中のAβ42の測定は、「Human β Amyloid(1-42) ELISA kit Woko, High Sensitive」（lot CAR2204, Fujifilm Wako 296-64401）の手順に従って測定した。　
１）100μLの抽出バッファーを細胞に添加し、混合。氷上で15 min 静置。
２）遠心分離（3,150×g、20 min）し、上清を回収。
３）上清50μLをキット添付のスタンダード希釈液50μLと混合（2倍希釈）。
４）キット添付の抗体（BAN50）固相化マイクロプレートにキット添付のスタンダード溶液の希釈系列（0.1、0.5、1、2、5、10、20 pmol/L）、10倍希釈したサンプル、キット添付のスタンダード希釈液（ブランク）をそれぞれ100μL/well で添加。
５）キット添付のプレートシールでシールし、冷蔵（4℃）で一晩反応。
６）ウェル内の液を除去し、300μL/well の１×キット添付の洗浄液で4回洗浄。
７）100μL/well のキット添付のHRP標識抗体（BC05）溶液を添加、冷蔵で1時間反応。
８）上記と同様に5回洗浄。
９）100μL/well のキット添付のTMB溶液を添加、室温で30分間反応（遮光）。
10）100μL/well のキット添付の停止液を添加。
11）450nmの吸光度を測定（30分以内）。各測定値からプランク測定値を減算し（プラン
出する。 (1-6) Measurement of A [beta] 42 measurements in each test sample of A [beta] 42 is measured according to the instructions in "Human β Amyloid (1-42) ELISA kit Woko, High Sensitive " (lot CAR2204, Fujifilm Wako 296-64401) did.
1) Add 100 μL of extraction buffer to cells and mix. Let stand on ice for 15 min.
2) Centrifuge (3,150 xg, 20 min) and collect the supernatant.
3) Mix 50 μL of the supernatant with 50 μL of the standard diluent supplied with the kit (2-fold dilution).
4) Dilution series (0.1, 0.5, 1, 2, 5, 10, 20 pmol/L) of the standard solution provided with the kit on the antibody (BAN50)-immobilized microplate provided with the kit, 10-fold diluted sample, and the kit provided Add 100 μL/well of each standard dilution (blank).
5) Seal with the plate seal attached to the kit and react overnight in the cold (4°C).
6) Remove the solution in the well and wash 4 times with 300 μL/well of the washing solution included in the 1x kit.
7) Add 100 μL/well HRP-labeled antibody (BC05) solution attached to the kit and incubate for 1 hour in refrigeration.
8) Wash 5 times as above.
9) Add 100 μL/well TMB solution attached to the kit and incubate at room temperature for 30 minutes (shielding).
10) Add 100 μL/well stop solution included in the kit.
11) Measure the absorbance at 450 nm (within 30 minutes). Planck measurement value is subtracted from each measurement value.
（１－７）試験結果
　上記の試験により、本試験条件において、以下のことが確認された。
１）細胞外のAβ42は細胞内に取り込まれるが、その濃度は高くない。（図８）
２）細胞外に試験ウイルスが存在する場合、細胞内のAβ42濃度が低下する傾向がみられる（図８）。
　これらのことは、細胞外のウイルスがAβ42と結合して細胞内への取り込みを抑制することを示唆し、Aβ42が脳内の自然免疫応答の産物で、脳内に侵入あるいは再活性化したウイルス等の病原体を捕捉する作用があるとの知見に合致した結果を示した。 (1-7) Test Results The above test confirmed the following under the test conditions.
1) Extracellular Aβ 42 is taken up into the cell, but its concentration is not high. (Figure 8)
2) When the test virus is present extracellularly, the intracellular Aβ 42 concentration tends to decrease (FIG. 8).
These things, suggesting that extracellular virus suppresses the uptake of bound to A [beta] 42 into the cell, the product of A [beta] 42 is the innate immune response in the brain, intrusion or reactivated in the brain The results are in agreement with the finding that they have an action of trapping pathogens such as viruses.
（実施例２）ホスカルネットによる、「ウイルス・Aβ複合体」に対する作用の確認
（２－１）ホスカルネットによる、「ウイルス・Aβ複合体」の細胞内取り込み抑制効果
　前記の試験サンプル用いて被験細胞内への「ウイルス・Aβ42複合体」の取り込み及び「ホスカビル（登録商標）」（ホスカルネットの静注製剤）がその取り込みを抑制する作用を有するか否かを、被験細胞内でのAβ42の濃度及びウイルスDNA数を測定する方法で検証した。
　試験に用いたSH－SY5Y細胞株について、Aβ42並びに各ウイルス溶液の添加48時間後に消化し、ウイルスGCをReal-time PCRにて測定した。その結果（図９）は以下の通リ。
１）試験細胞に対し、遺伝子検査において、HHV-6A、HHV-6B及びHHV-7は単独でも感染するが、HHV-6A及びHHV-7、又はHHV-6BとHHV-7との複合感染も観察された。
２）HHV-6AとHHV-7とは、双方に高い濃度のGCがみられ感染の相性はいいと考えられる。他方、HHV-7の存在下ではHHV-6BのGCが著しく低く検出された。この背景にはHHV-6Bの試験細胞への感染力が低いことが示唆された。
３）培地にホスカルネットを添加した群では、Aβ42の量が多くなるにつれてHHV-6のGCが減少する傾向がみられた。しかし、ホスカルネット無添加群ではそのような傾向はみられなかった。このことは、Aβ42の存在下においてもホスカルネットがHHV-6に対する抗ウイルス効果を有することを示唆している。
　試験の結果、ホスカビル（登録商標）の添加による被験細胞内でのウイルスDNA数の減少効果は示されたが、それがホスカビル（登録商標）の抗ウイルス作用によるか、あるいは「ウイルス・Aβ複合体」の細胞内への取り込みの抑制によるかは、本試験条件では確認できなかった。
　また、ホスカビル（登録商標）添加群では、細胞内HHV-6のDNA数は添加Aβの増加に伴って減少していた。このことは、試験条件下において細胞外のAβ42はウイルスと共に被験細胞内に取り込まれていること、Aβ42はウイルスの細胞内取り込みを抑制すること、ホスカビル（登録商標）の添加はかかる抑制を阻害しないことを示した。
　上記データは、細胞外に出現したウイルスなどの病原体を捕捉するというAβの自然免疫作用（W Eimer et al., Neuron 99 (1): 56-63, 2018、RD Moir et al., Alzheimer’s & Dementias 14 (2018): 1602-1614, 201）がHHV-6/7感染にも適用されることを裏付ける証左といえた。 (Example 2) Confirmation of action of foscarnet on "virus/Aβ complex" (2-1) Effect of foscarnet on intracellular uptake of "virus/Aβ complex" Using the test sample In the test cells, it was determined whether the "virus/Aβ 42 complex" was taken up into the test cells and whether "foscabir (registered trademark)" (foscarnet intravenous preparation) had an action of suppressing the uptake. It was verified by a method of measuring the concentration of Aβ 42 and the number of viral DNA.
The SH-SY5Y cell line used in the test was digested 48 hours after the addition of Aβ 42 and each virus solution, and the virus GC was measured by Real-time PCR. The result (Fig. 9) is as follows.
1) HHV-6A, HHV-6B and HHV-7 can infect test cells by genetic test alone, but also HHV-6A and HHV-7 or combined infection of HHV-6B and HHV-7. Was observed.
2) HHV-6A and HHV-7 have high concentrations of GC in both and are considered to be compatible with each other. On the other hand, in the presence of HHV-7, the GC of HHV-6B was remarkably low. Against this background, it was suggested that HHV-6B had low infectivity to test cells.
3) In the group in which foscarnet was added to the medium, the GC of HHV-6 tended to decrease as the amount of Aβ 42 increased. However, no such tendency was observed in the foscarnet-free group. This suggests that foscarnet has an antiviral effect on HHV-6 even in the presence of Aβ 42 .
As a result of the test, the effect of reducing the number of viral DNA in the test cells by the addition of foscavir (registered trademark) was shown to be due to the antiviral effect of foscavir (registered trademark) or the "virus-Aβ complex". It could not be confirmed under the present test conditions whether it was due to the suppression of the intracellular uptake of "".
In the foscavir (registered trademark)-added group, the intracellular HHV-6 DNA number decreased with an increase in the added Aβ. This means that extracellular Aβ 42 is taken up into the test cells together with the virus under the test conditions, Aβ 42 suppresses the intracellular uptake of the virus, and addition of foscavir (registered trademark) suppresses such suppression. It was shown not to inhibit.
The above data show the innate immune action of Aβ that captures extracellular pathogens such as viruses (W Eimer et al., Neuron 99 (1): 56-63, 2018, RD Moir et al., Alzheimer's & Dementias. 14 (2018): 1602-1614, 201) can be said to be proof that it also applies to HHV-6/7 infection.
（２－２）ホスカルネットによる、Aβ存在下でのHHV-6/HHV-7の細胞障害性に対する影響
　Aβの存在下において、HHV-6A、HHV6-B及びHHV-7の細胞障害性を検証する為、被験細胞として、神経芽細胞腫・SH-SY5Y株に各ウイルス溶液を添加してウイルス感染させ、24時間及び48時間後の細胞数を測定した。また、ホスカビル（登録商標）の添加が細胞数にどのような影響を及ぼすかを調べた。
　被験細胞の調製及びAβ42の添加とウイルス感染の手順は上記と同じであり、結果は以下の通り。
１）上記試験条件下において、Aβ42には細胞傷害性はなく、48時間後には24時間後に比べて細胞が30％以上増加した（図７）。しかし、培地へのAβ42添加と無添加群との間で大きな差は生じていない。
２）試験条件下(800μM)において、ホスカビル（登録商標）は細胞増殖を抑制(無添加群と比較して84％)することを示した。
３）各ウイルス添加群でも細胞数は増えており、Aβ42のウイルスの細胞障害性は低いことを示唆している。
４）ウイルス存在下でのAβ42無添加群とAβ42添加群とでは、Aβ42添加群の方が細胞数は増えている。しかし、Aβ42の1000nM 添加群では細胞数は24時間後計計測時と比べて維持か低下傾向にある。
　以上の結果は、試験条件下ではAβ42の細胞毒性は観察されず、むしろウイルス存在下においても細胞増殖が行われていることから、Aβ42にはウイルスから細胞を守る作用があることが、HHV-6/7についても実証できたこと示唆している。 (2-2) Effect of HOSV-6 on the cytotoxicity of HHV-6/HHV-7 in the presence of Aβ For verification, as test cells, each virus solution was added to the neuroblastoma SH-SY5Y strain to cause virus infection, and the number of cells was measured after 24 hours and 48 hours. In addition, it was examined how the addition of foscavir (registered trademark) affects the cell number.
The procedure for preparation of test cells, addition of Aβ 42 , and virus infection was the same as above, and the results are as follows.
1) Under the above test conditions, Aβ 42 had no cytotoxicity, and cells increased by 30% or more after 48 hours as compared with after 24 hours (FIG. 7). However, there is no significant difference between the Aβ 42 addition to the medium and the non-addition group.
2) It was shown that under the test conditions (800 μM), foscavir (registered trademark) suppresses cell growth (84% compared to the non-addition group).
3) The number of cells increased in each virus-added group, suggesting that Aβ 42 virus has low cytotoxicity.
4) In the Aβ 42 addition group and the Aβ 42 addition group in the presence of virus, the number of cells increased in the Aβ 42 addition group. However, the number of cells in the Aβ 42- added group of 1000 nM tended to be maintained or decreased compared to the time measured 24 hours later.
The above results indicate that Aβ 42 has no cytotoxicity under the test conditions, and rather, that cell proliferation is performed even in the presence of virus, Aβ 42 has an action of protecting cells from the virus. It also suggests that HHV-6/7 could be demonstrated.
（２－３）Aβ存在下でのホスカルネットの抗ウイルス作用
　ホスカルネットの抗ウイルス効果を調べたいくつかの公知文献はある。しかし、アミロイドβ存在下でのHHV-6及びHHV-7に対するホスカルネットの効果を検討した試験は知られていない。
　そこで、Aβ42の存在下においてホスカルネットのHHV-6A、HHV-6B及びHHV-7に対する抗ウイルス作用を、神経芽細胞SH-SY5Y株を用い、細胞中のウイルスDNA（GC）をReal-time PCRにて解析する方法で検証した。
　その結果は、ホスカビル（登録商標）の試験液(800μM)溶液を添加した群で、細胞外のAβ４２の量が多くなるにつれてHHV-6の細胞内ゲノム数が減少した。しかし、ホスカビル（登録商標）の未添加群ではそのような減少傾向はみられず、Aβ42存在下でのホスカビル（登録商標）のHHV-6/7に対する抗ウイルス効果が示唆された（図９）。つまり、ホスカビル（登録商標）の抗ウイルス作用の結果、細胞内のウイルスあるいは感染細胞の減少を示唆した。この細胞内ウイルスの減少はウイルス感染に伴う細胞死の抑制が期待できる。 (2-3) Antiviral action of foscarnet in the presence of Aβ There are several known documents examining the antiviral effect of foscarnet. However, there are no known studies examining the effect of foscarnet on HHV-6 and HHV-7 in the presence of amyloid β.
Therefore, in the presence of Aβ 42, the antiviral action of foscarnet against HHV-6A, HHV-6B and HHV-7 was investigated by using neuroblast SH-SY5Y strain, and the viral DNA (GC) in the cells was Real- It was verified by the method of analysis by time PCR.
As a result, in the group to which the test solution (800 μM) of foscavir (registered trademark) was added, the intracellular genome number of HHV-6 decreased as the amount of extracellular Aβ 42 increased. However, such a decreasing tendency was not observed in the foscavir (registered trademark)-free group, suggesting the antiviral effect of foscavir (registered trademark) on HHV-6/7 in the presence of Aβ 42 (FIG. 9). ). That is, it was suggested that the antiviral effect of foscavir (registered trademark) resulted in a decrease in intracellular virus or infected cells. This decrease in intracellular virus can be expected to suppress cell death associated with virus infection.
（実施例３）アミロイドβ（オリゴマーを含む）の存在下において、ホスカルネットの神経細胞へのカルシウムの取り込み抑制効果
　96穴マイクロプレート上でSH-SY5Y細胞を培養した後、培養液にAβのみ、及びAβとホスカルネットを添加し、さらに1～3日間培養する。細胞を傷つけないように培養液を取り除き洗浄後、蛍光基質液を添加して、蛍光強度測定により細胞内カルシウム量を計測する。
　前記（参考例６）の結果を参酌すれば、ホスカルネットを添加した細胞では、Aβ及び／又はAβオリゴマー取り込みによる細胞内カルシウム濃度の上昇が抑制され、細胞内カルシウム濃度の恒常性が保たれることが推認できる。 (Example 3) In the presence of amyloid β (including oligomer), foscarnet inhibits calcium uptake into nerve cells After culturing SH-SY5Y cells on a 96-well microplate, only Aβ was added to the culture solution. , And Aβ and foscarnet are added, and the cells are further cultured for 1 to 3 days. After removing the culture solution so as not to damage the cells and washing, a fluorescent substrate solution is added and the intracellular calcium amount is measured by fluorescence intensity measurement.
In consideration of the results of (Reference Example 6), in cells to which foscarnet was added, the increase in intracellular calcium concentration due to Aβ and/or Aβ oligomer uptake was suppressed, and the homeostasis of intracellular calcium concentration was maintained. Can be inferred.
（実施例４）ホスカルネットの抗βセクレターゼ作用
　ホスカルネットは抗金属（亜鉛）酵素阻害活性を有することから、アスパラギン酸プロテアーゼ（活性部位に亜鉛を有する）であるβセクレターゼの活性を阻害する作用を有するか否かを以下の試験を用いて検証した。
１）試験材料として、ホスカビル（登録商標）（点滴静注用ホスカビル（登録商標）注24mg/mL, 製造番号XX26。主成分としてホスカルネット6gを含む）、ANASPEC社製「SensoLyte 520 BASE1 Assay Kit FluorimetricTM」（型番AS-71144）、同キット付属の陽性対照阻害剤・LY2886721、市販されているCEM社の阻害剤ADZ3839（free base）(CS-5933)を用いた。
２）試験方法
　ホスカビル（登録商標）について、0.19 Mをキット付属のバッファーで190倍希釈を行い1mMの被験溶液を調整し、最終濃度100μM、10μM、1μM、10nM、1nMの5検体を調製した。
　キット付属の阻害剤・LY2886721は250nM,市販の阻害剤AZD3839 は10μMの検体を調製し、キットの試験プロトコールに従って試験を実施した。
３）結果
　ホスカビル（登録商標）のいずれの検体も、試験条件においては、βセクレターゼ阻害効果は殆ど見られなかった（図６）。このことは、ホスカルネットはADの治療において、Aβではなく、脳内に侵入したHHV-6/ 7、又は再活性化した潜伏HHV-6/7を直接の治療標的とするものであり、結果としてウイルス病原体への自己免疫応答として形成されるAβの形成を抑える作用を有することを示す。 (Example 4) Anti-β secretase action of foscarnet Since foscarnet has antimetal (zinc) enzyme inhibitory activity, it inhibits the activity of β-secretase, which is an aspartic protease (having zinc at the active site). Whether or not it has an effect was verified using the following test.
1) As a test material, foscavir (registered trademark) (foscavir (registered trademark) for instillation, 24 mg/mL, serial number XX26, containing 6 g of foscarnet as the main component), "SensoLyte 520 BASE1 Assay Kit by ANASPEC" Fluorimetric ™ ” (model number AS-71144), the positive control inhibitor LY2886721 attached to the kit, and the commercially available CEM inhibitor ADZ3839 (free base) (CS-5933) were used.
2) Test method For Hoscavir (registered trademark), 0.19 M was diluted 190-fold with a buffer included in the kit to prepare a test solution of 1 mM, and 5 test samples having final concentrations of 100 μM, 10 μM, 1 μM, 10 nM, and 1 nM were prepared.
The inhibitors included in the kit, LY2886721, 250 nM, and the commercially available inhibitor AZD3839, 10 μM, were prepared as samples, and the test was performed according to the test protocol of the kit.
3) Results Almost no sample of foscavir (registered trademark) showed almost no β-secretase inhibitory effect under the test conditions (Fig. 6). This indicates that foscarnet, in the treatment of AD, is not Aβ, but HHV-6/7 that has invaded the brain or reactivated latent HHV-6/7 is a direct therapeutic target. As a result, it shows that it has an action of suppressing the formation of Aβ formed as an autoimmune response to a viral pathogen.
（実施例５）HHV-６/７の感染細胞に対する殺作用/効果
　非感染細胞（HHV-6Aに関してはHSB-2、HHV-6B及びHHV-7に関してはSup-T1）に上記手順により得られたウイルス溶液を添加し、ウイルス感染の有無、感染細胞の形態の変化を以下により観察した。
（５－１）HHV-6Aに関して
　HSB-2（未感染株）のみを培養する培地と、未感染株を培養する培地に前記手順により得られたHHV-6Aウイルス溶液を添加し、各培地からサンプリングし、光学顕微鏡（Olympus CKX 41）にて各培地中の細胞の形態を観察した。
　その結果は図１０－１（未感染細胞）では、各細胞の形や大きさは均一で、死亡した細胞は殆ど観察されていない。しかし、図１０－２（HHV-6A添加）では、肥大した細胞及び死細胞が散見している。 (Example 5) Killing/effect of HHV-6/7 on infected cells Obtained by the above procedure on non-infected cells (HSB-2 for HHV-6A, Sup-T1 for HHV-6B and HHV-7) The virus solution was added, and the presence or absence of virus infection and changes in the morphology of infected cells were observed as follows.
(5-1) Regarding HHV-6A, a medium for culturing only HSB-2 (uninfected strain) and a medium for culturing uninfected strain are added with the HHV-6A virus solution obtained by the above procedure, Sampling was performed and the morphology of cells in each medium was observed with an optical microscope (Olympus CKX 41).
As a result, in FIG. 10-1 (uninfected cells), the shape and size of each cell were uniform, and almost no dead cells were observed. However, in FIG. 10-2 (HHV-6A added), enlarged cells and dead cells are scattered.
（５－２）HHV-6B及びHHV-7に関して
　SupT-1（未感染株）のみを培養する培地と、未感染株を培養する培地に前記手順により得られたHHV-6Bウイルス溶液又はHHV-7ウイルス溶液を添加し、サンプリングした培地を光学顕微鏡（Olympus CKX 41）にて20倍で各培地中の細胞の形態を観察し、Leica MC 120HDにて露光モード自動、解像度HD1080－50にて撮影した。
　その結果は、図１１－１（ウイルス未感染細胞）では、死細胞は見られず、細胞の形、大きさも均一である。しかし、図１１－２（HHV-B添加）及び図１２（HHV-7 添加）で僅かな死細胞の他に、細胞表面がゴツゴツした細胞、肥大した細胞が散見される。 (5-2) Regarding HHV-6B and HHV-7, a medium for culturing only SupT-1 (uninfected strain) and a medium for culturing an uninfected strain are used in the HHV-6B virus solution or HHV- 7 The virus solution was added, and the sampled medium was observed with an optical microscope (Olympus CKX 41) 20 times to observe the morphology of the cells in each medium, and the Leica MC 120 HD was used for automatic exposure mode and taken at a resolution of HD1080-50. did.
As a result, in FIG. 11-1 (cells not infected with virus), dead cells were not seen, and the cell shape and size were uniform. However, in addition to a few dead cells in FIG. 11-2 (HHV-B added) and FIG. 12 (HHV-7 added), cells with rugged cell surface and enlarged cells are scattered.
（５－３）免疫染色法による試験細胞のウイルス感染の確認
　培地中の未感染のHSB-2及び Sup-T1に前記手順により得られた各ウイルス溶液を添加した場合、未感染細胞がウイルスに感染するか否かについて、HHVを構成するタンパク質に対する抗体を用いた免疫染色試験を以下の手順で行い、検証した。
１）HSB-2、Sup-T1を7.6×10４ cells/0.25 mL/well (3.3 ×105 cell/mL)で24-well plateへ播種。一晩培養。
２）上記手順により得られたHHV-6A、HHV-6B及びHHV-7の各ウイルス溶液を、それぞれMOI 1.0 となるよう添加。48時間培養。
３）各細胞を含む培地全量を1.5 mL チューブに移す。
４）ウイルスを含む培地を除去し、それぞれについてPBS（－）(lot.TWK7042,Fujifilm Wako 166-23556)で2回洗浄。
５）各細胞に氷冷したアセトンを添加し、氷上で10分間保温。
６）アセトンを除去し、0.5％ BSA含有PSB(－)で3回洗浄。
７）0.5％ BSA含有FBS(－)で200倍希釈した一次抗体を添加し、4℃で一晩保温。一次抗体を含む液を除去し、0.5％ BSA含有PBS（－）で3回洗浄。
　本実験の免疫染色に使用した一次抗体は、下記（表１３）の通りである。 (5-3) Confirmation of virus infection of test cells by immunostaining When each virus solution obtained by the above procedure was added to uninfected HSB-2 and Sup-T1 in the medium, uninfected cells became viral. Whether to be infected or not was verified by performing an immunostaining test using an antibody against a protein constituting HHV according to the following procedure.
1) Seed HSB-2 and Sup-T1 in 24-well plate with 7.6 × 10 4 cells/0.25 mL/well (3.3 × 10 5 cells/mL). Overnight culture.
2) Add each virus solution of HHV-6A, HHV-6B and HHV-7 obtained by the above procedure so that the MOI becomes 1.0. Culture for 48 hours.
3) Transfer the entire medium containing each cell to a 1.5 mL tube.
4) The medium containing the virus was removed, and each was washed twice with PBS(-) (lot.TWK7042, Fujifilm Wako 166-23556).
5) Add ice-cold acetone to each cell and incubate on ice for 10 minutes.
6) Remove acetone and wash 3 times with PSB(-) containing 0.5% BSA.
7) Add primary antibody diluted 200 times with FBS(-) containing 0.5% BSA and incubate at 4°C overnight. Remove the solution 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 shown below (Table 13).
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
８）5倍希釈した2次抗体（TaKaRa POD conjugate anti-mouse, for tissue, lot AE 45500, Takara MK 204）を各細胞に添加し、室温で1時間保温。
９）0.5％ BSA含有FBS(－)で3回洗浄。
１０）DAB発色液（TaKaRa DAB Substrate,lot AI2P021, Takara MK 210）を添加し、室温で5分間発色。
１１）各細胞にDAB発色液を除去し、PBS(－)で洗浄し、検鏡（Olympus CKX 41）
　HHVタンパク（各ウイルス溶液）を添加していない未感染のHSB-2及び Sup-T1細胞では抗体による陽性反応（DABによる茶褐色の染色）がみられなかった（図１３－１，１３－２，１３－３）。他方、ウイルス溶液を添加した細胞群では、多くの細胞で抗体による陽性反応がみられた（図１３－４，１３－５，１３－６）。
　これによって、試験に用いた各未感染細胞は試験に用いたウイルス（HHV-6A、HHV-6B及びHHV-7）に感染することが確認された。また、感染細胞を示す染色部分は、正常な形態を示す細胞まで及んでいることはウイルス感染下でも生存する細胞の存在を示唆した。尚、試験に用いた各細胞は試験ウイルスに対する感染率が高いことを示した。 8) Add 5 times diluted secondary antibody (TaKaRa POD conjugate anti-mouse, for tissue, lot AE 45500, Takara MK 204) to each cell and incubate at room temperature for 1 hour.
9) Wash 3 times with FBS(-) containing 0.5% BSA.
10) Add DAB color development solution (TaKaRa DAB Substrate, lot AI2P021, Takara MK 210) and develop color for 5 minutes at room temperature.
11) Remove the DAB color development solution from each cell, wash with PBS(-), and examine with a microscope (Olympus CKX 41).
No positive reaction with antibodies (dark brown staining with DAB) was observed in uninfected HSB-2 and Sup-T1 cells to which HHV protein (each virus solution) was not added (FIGS. 13-1, 13-2, 13-3). On the other hand, in the cell group to which the virus solution was added, a positive reaction due to the antibody was observed in many cells (Figs. 13-4, 13-5, 13-6).
From this, it was confirmed that each uninfected cell used in the test was infected with the viruses (HHV-6A, HHV-6B and HHV-7) used in the test. In addition, the stained portion showing infected cells extended to cells showing normal morphology, suggesting the existence of cells that survived virus infection. Each cell used in the test was shown to have a high infection rate for the test virus.
（５－４）試験結果
　上記試験によって、HHV-6A、HHV-6B、HHV-7に感染した細胞には、細胞表面に凹凸が生じるもの、肥大化するという形態変化が起きることが明らかになった。この内、細胞表面に凹凸が生じた細胞は、その径が非感染細胞と変わらないこと、アポトーシスの場合は細胞膜の形質変化を起点とすることから、アポトーシス細胞を示している。
他方、細胞の肥大化はネクローシスの典型的な形態（参考文献１５）であることから、肥大化した細胞は、ネクローシスする細胞と考えられる。また、免疫染色で陽性反応を示す細胞は多いが、アポトーシス及びネクローシスとなる細胞数が少ないことから、感染細胞の多くは正常な状態（形態）を示すことが明らかになった。
　これまで、ウイルスに感染した細胞はアポトーシスすると考えられていたが（非特許文献１０、１１、参考文献３０，３１，３７，３８）、上記の試験により、HHV-6A、HHV-6B及びHHV-7に感染した細胞はアポトーシスだけでなく、ネクローシスをも惹起することが初めて明らかにされた。
　以上の知見は、ADにおいて脳内、特に神経細胞の新生が行われている海馬において神経細胞やグリア細胞に潜伏感染しているHHV-6AやHHV-6Bが宿主細胞のアポトーシスを含む細胞死に際して再活性化した場合、炎症を生起した部位に遊走したT細胞がネクローシスをも惹起し、当該細胞からDAMPs（Damage/ Danger Associated Molecular Patterns）として様々な炎症起因物質（例えばHMGB-1，TDP-43、GSK-3β、グルタミン酸、Ca＋＋等）が放出されて炎症が増強し、それに伴う様々な病理、特に神経細胞の過剰な死を招くことを示唆している（非特許文献１８及びQ Chen et al., Signal Transduction and Targeted Therapy, 3(18): 1-11, 2018）。 (5-4) Test Results The above test revealed that cells infected with HHV-6A, HHV-6B, and HHV-7 undergo morphological changes such as cell surface unevenness and hypertrophy. It was Among these, cells having irregularities on the cell surface are apoptotic cells because their diameter is the same as that of non-infected cells and in the case of apoptosis, the origin is a change in the cell membrane.
On the other hand, since hypertrophy of cells is a typical form of necrosis (Reference Document 15), hypertrophied cells are considered to be necrotic cells. In addition, although many cells showed a positive reaction by immunostaining, the number of cells that became apoptotic and necrotic was small, which revealed that many of the infected cells showed a normal state (morphology).
Until now, it has been considered that cells infected with a virus undergo apoptosis (Non-patent Documents 10, 11 and References 30, 31, 37, 38), but by the above test, HHV-6A, HHV-6B and HHV-. It was revealed for the first time that cells infected with 7 induce necrosis as well as apoptosis.
The above findings suggest that HHV-6A and HHV-6B, which are latently infected with nerve cells and glial cells in the hippocampus in which the neuron is newly formed in AD, undergo cell death including apoptosis of host cells. When reactivated, T cells that have migrated to the site of inflammation also induce necrosis, and various inflammatory substances (eg, HMGB-1, TDP-43 as DAMPs (Damage/ Danger Associated Molecular Patterns)) are generated from the cells. , GSK-3β, glutamic acid, Ca ++, etc. are released to enhance inflammation, which leads to various pathologies associated therewith, especially 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)

  1. 　アルツハイマー病（「AD」）を治療または予防する為の医薬組成物であって、ヒトヘルペスウイルス６型A及び６型B（以下、併せて「HHV-6」という）及び７型（「HHV-7」）に対する抗ウイルス活性を持つ化合物を有効成分とし、薬学的に許容される担体を含む医薬組成物。 A pharmaceutical composition for treating or preventing Alzheimer's disease (“AD”), which comprises human herpesvirus type 6 A and type 6 B (hereinafter collectively referred to as “HHV-6”) and type 7 (“HHV- A pharmaceutical composition comprising a compound having antiviral activity against 7") as an active ingredient and a pharmaceutically acceptable carrier.
  2. 　前記化合物が、更に単純ヘルペスウイルス1型（「HSV-1」）及びサイトメガロウイルス（「CMV」）に対する抗ウイルス活性を有する化合物である、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the compound further has an antiviral activity against herpes simplex virus type 1 (“HSV-1”) and cytomegalovirus (“CMV”).
  3. 　前記化合物が、標的ウイルス由来のDNAポリメラーゼのピロリン酸結合部位に結合し、選択的にウイルスの増殖を阻害する化合物でである、請求項１又は２に記載の医薬組成物。 3. The pharmaceutical composition according to claim 1 or 2, wherein the compound is a compound that binds to a pyrophosphate binding site of a DNA polymerase derived from a target virus and selectively inhibits virus growth.
  4. 　前記化合物が、以下の基本骨格（式１）で表されるホスホノ酢酸もしくはホスホノギ酸又はそれらの誘導体であるピロリン酸アナログである、請求項１～３のいずれかに記載の医薬組成物。
    Figure JPOXMLDOC01-appb-C000001
         4. The pharmaceutical composition according to claim 1, wherein the compound is a phosphonoacetic acid or phosphonoformic acid represented by the following basic skeleton (formula 1) or a pyrophosphate analog which is a derivative thereof.
    Figure JPOXMLDOC01-appb-C000001
  5. 　前記化合物が、下記（式２）で表されるホスギノ酸3ナトリウムの6水和物（以下「ホスカルネット」という）であり、（JNN）／INN表記でFoscarnet Sodium と表記される、請求項１～４のいずれかに記載の医薬組成物。
    Figure JPOXMLDOC01-appb-C000002
     （式２）     The compound is trisodium phosphinate hexahydrate represented by the following (formula 2) (hereinafter referred to as "foscarnet"), and is represented by Foscarnet Sodium in (JNN)/INN notation. 5. The pharmaceutical composition according to any one of 1 to 4.
    Figure JPOXMLDOC01-appb-C000002
     (Formula 2)
  6. 　ガンクロシクロビル、バラシクロビル、ペンシクロビル、ブリブジンからなる群から選択された少なくとも1つのヌクレオシド類縁体；シドフォビル、そのプロドラッグもしく誘導体又は他のヌクレオタイド類縁体の製剤；ヌクレオシド化合物の製剤；非ヌクレオシドDNAポリメラーゼ抑制化合物の製剤；アメナメビルもしくは他のヘリカーゼ・プライマーゼ阻害化合物の製剤；ウイルスDNAカプシドへのパッケージ阻害化合物；レテルモビルもしくは他のウイルスDNAターミナーゼ複合体の阻害剤；ニボルマブもしくは他のPD-1抗体、PD-1受容体抗体を含有する製剤の群から選択された少なくとも1つの製剤と併用して投与されることを特徴とする、請求項５に記載の医薬組成物。 At least one nucleoside analog selected from the group consisting of ganclocyclovir, valacyclovir, penciclovir, brivudine; preparation of cidofovir, its prodrug or derivative or other nucleotide analogs; preparation of nucleoside compounds; non-nucleoside DNA polymerase Formulation of inhibitory compounds; Formulation of amenamevir or other helicase primase inhibitor compounds; Package inhibitor compounds to viral DNA capsids; Inhibitors of letermovir or other viral DNA terminase complexes; Nivolumab or other PD-1 antibodies, PD The pharmaceutical composition according to claim 5, which is administered in combination with at least one preparation selected from the group of preparations containing -1 receptor antibody.
  7. 　ドネペジル、ガランタミン、リバスチグミン、もしくはメマンチン製剤、神経保護剤、神経伝達物質からなる群から選択された少なくとも1つのAD治療剤又は神経伝達物質の移動の調整剤；アミロイドβもしくはAβオリゴマーを標的とする薬剤；タウタンパク質を標的とする薬剤；細胞への過剰なカルシウム流入抑制剤；ワクチン、イブプロフェン、ギンコエキス、もしくは他の抗炎症剤；インスリンもしくは他の抗糖尿病薬；抗IL-6抗体、抗IL-12抗体、もしくは他の炎症性サイトカイン、ケモカインの中和剤の群から選択された少なくとも1つの製剤と併用して投与されることを特徴とする、請求項５に記載の医薬組成物。 Donepezil, galantamine, rivastigmine, or at least one AD therapeutic agent or neurotransmitter migration regulator selected from the group consisting of memantine preparations, neuroprotective agents, and neurotransmitters; agents that target amyloid β or Aβ oligomers Agents targeting tau protein; inhibitors of excessive calcium influx into cells; vaccines, ibuprofen, ginkgo extracts, or other anti-inflammatory agents; insulin or other antidiabetic agents; anti-IL-6 antibodies, anti-IL- The pharmaceutical composition according to claim 5, which is administered in combination with at least one preparation selected from the group consisting of 12 antibody, or other inflammatory cytokine and a chemokine neutralizing agent.
  8. 　ADに罹患するリスク又はADの進展状況を診断するための薬剤組成物であって、HHV-6A、HHV-6B及び／又はHHV-7に由来するDNAもしくはタンパク質、又はHHV-6A、HHV-6B及び／又はHHV-7に特異的な抗体もしくはその断片を、ADバイオマーカーとして検出可能な化合物を含む、薬剤組成物。 A pharmaceutical composition for diagnosing the risk of suffering from AD or the progress of AD, wherein HHV-6A, HHV-6B and/or DNA or protein derived from HHV-7, or HHV-6A, HHV-6B And/or a pharmaceutical composition comprising a compound capable of detecting an antibody or a fragment thereof specific for HHV-7 as an AD biomarker.
  9. 　前記薬剤組成物が、被験者の脳脊髄液（CSF）、血漿、血液、又は唾液中に存在するHHV-6A、HHV-6B及び／又はHHV-7に由来するDNAもしくはタンパク質、又はHHV-6A、HHV-6B及び／又はHHV-7に特異的な抗体もしくはその断片と結合する化合物を含む、請求項８に記載の薬剤組成物。 The pharmaceutical composition, cerebrospinal fluid (CSF) of the subject, plasma, blood, or HHV-6A present in saliva, HHV-6B and / or DNA or protein derived from HHV-7, or HHV-6A, The pharmaceutical composition according to claim 8, which comprises a compound that binds to an antibody or a fragment thereof specific for HHV-6B and/or HHV-7.
  10. 　ADの治療に用いる抗ウイルス剤の治療効果を確認するための薬剤組成物であって、HHV-6A、HHV-6B及び／又はHHV-7に由来するウイルスDNAもしくはウイルスタンパク質、又はHHV-6A、HHV-6B及び／又はHHV-7に特異的な抗体もしくはその断片を、ADバイオマーカーとして検出可能な化合物を含む、薬剤組成物。 A pharmaceutical composition for confirming the therapeutic effect of an antiviral agent used for treating AD, comprising HHV-6A, HHV-6B and/or viral DNA or protein derived from HHV-7, or HHV-6A, A pharmaceutical composition comprising a compound capable of detecting an antibody or a fragment thereof specific to HHV-6B and/or HHV-7 as an AD biomarker.
  11. 　前記薬剤組成物が、被験者の脳脊髄液（CSF）、血漿、血液、又は唾液中に存在するHHV-6A、HHV-6B及び／又はHHV-7に由来するDNAもしくはタンパク質、又はHHV-6A、HHV-6B及び／又はHHV-7に特異的な抗体もしくはその断片と結合する化合物を含む、請求項１０に記載の薬剤組成物。 The pharmaceutical composition, cerebrospinal fluid (CSF) of the subject, plasma, blood, or HHV-6A present in saliva, HHV-6B and / or DNA or protein derived from HHV-7, or HHV-6A, The pharmaceutical composition according to claim 10, comprising a compound that binds to an antibody or a fragment thereof specific for HHV-6B and/or HHV-7.
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Non-Patent Citations (7)

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EIMER WILLIAM A., VIJAYA KUMAR DEEPAK KUMAR, NAVALPUR SHANMUGAM NANDA KUMAR, RODRIGUEZ ALEX S., MITCHELL TERYN, WASHICOSKY KEVIN J: "Alzheimer's disease- associated beta-amyloid is rapidly seeded by Herpesviridae to protect against brain infection", NEURON, vol. 99, 11 July 2018 (2018-07-11), pages 56 - 63, XP055721798 *
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