No. 224

Prevention and Medication for Lifestyle Diseases Related to High Mobility Group Box-1（HMGB1）and its Receptor, RAGE Masahiro Nishibori Professor Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Okayama University Background In 1999, as a result of their search for a late mediator of sepsis, the American research group of Kevin Tracey discovered that a factor with a molecular weight of 28kDa was secreted into the macrophage growth supernatant 8 hours after LPS stimulation. This factor was identified as the intranuclear chromatin binding factor high mobility group box-1（HMGB1） （Wan et al., Science 1999）. Tracey et. al. proved that anti-HMBG1 polyclonal antibodies improved survival rate in a mouse LPS lethality model, but the observation that an intranuclear factor could be a lethality mediator was a big surprise. Overall seeds The research team representative, Masahiro Nishibori, has been involved in research on regulating the activity of macrophages for quite a while. After discovering a large amount of HMGB1 adhering to the inside of an LPS absorbing column （Toraymyxin） , which is used as an extracorporeal circulation column for sepsis（Nishibori, 2005）, Nishibori began to study the dynamics of HMGB1 in various inflammation models and to evaluate the treatment of diseases with anti-HMGB1 monoclonal antibodies（Liu et al., FASEB J, 2007）. In particular, in a rat brain infarction model in which the middle cerebral artery was blocked for two hours and then allowed to flow again, from the early acute phase of cerebral ischemia, the extracellular release of HMGB1 that was inside the nuclei of nerve cells was observed with a confocal laser microscope. There have been various reports of cerebral cytokines and inflammation-related molecules during the acute phase of ischemic stroke, but changes observed one hour after the stroke are the earliest observed changes. In other words, the release of HMGB1 is understood to be the quickest of the various physiological warnings that are activated. Administration of anti-HMGB1 monoclonal antibody created by the researcher significantly inhibited the destruction of both the form and function of the blood-brain barrier as well as inhibiting the production of related inflammation-related molecules iNOS MMP-9 and TNF- α . As a result, the size of the brain infarction that formed 24-48 hours later was dramatically reduced, and a large improvement in motor paralysis symptoms was observed. －  131  －

This discovery/invention has great significance, since it could lead to the development of a treatment for the acute phase of ischemic stroke that is second only to tissue plasminogen activator（t-PA） . This has been evaluated highly, and Patent Application 1（brain infarction inhibitor, patent no. 3876325, WO2007/049468）was awarded the 21st Century Invention Promotion Award（Fig. 2 in last page）among the 2009 National Commendations for Invention（Japan Institute of Invention and Innovation; President: Prince Hitachi） . Around the same time, this treatment method was observed to be effective for cerebrovascular spasms following subarachnoid hemorrhages, which has long been a problem in the brain surgery field, and Patent Application 2 for this was awarded patent rights（cerebrovascular spasm inhibitor, patent no. 3882090, WO2007/135992） . In this way, the research was evaluated at the same time as being effective for pathological states in which HMGB1 was not originally thought to be involved, since HMGB1 targeting antigen was found to be involved. Artherosclerosis I will now explain about research seeds dealing with arterial sclerosis one of the causes of hypertension, and in particular artherosclerosis. In recent estimates by the Ministry of Health, Labor and Welfare, 30 % percent of the adult population（ages 20-59）is suspected to have metabolic syndrome, and that percentage is expected to rise in the near future. With metabolic syndrome, the risk of developing hypertension and diabetes increases, and a significant progression of artherosclerosis invites the development of serious diseases（Fig. 1） . It does without saying that it is important to raise awareness of prevention by the general population, but there are limits to an individual’s ability to significantly change eating habits and lifestyles. On the other hand, in the medical field, an effective treatment method for treating artherosclerosis, which tends to develop unnoticed, has yet to be found. T h e r e h a s b e e n n o significant development in treatment methods since the appearance of LDL（Fig. 1）

cholesterol lowering statins.

Artherosclerosis is a vascular disease that invariably accompanies lifestyle diseases（metabolic syndrome）. Often, it develops unnoticed, so that even the patient is not aware of it. Nishibori measured the HMGB1 levels in the blood of occlusive arterial disease patients and found a strong correlation －  132  －

between progression of the disease and the amount of HMGB1 in the blood. Considering this together with reports that RAGE, one of the HMGB1 receptors, is highly expressed in regions of artherosclerosis, a theory was formulated that macrophage productivity HMGB1 and its RAGE receptor mediate inflammation amplification and monocyte incursions in the intimal layer of blood vessels. First, the high expression of HMGB1 protein in locations of artherosclerosis generated in ApoE -/- mice by feeding them a high fat diet was confirmed by immunohistological staining（Fig. 3）. The accumulation of monocytes in the plaque, expression of ICAM-1 and VCAM-1 in the endothelial cells, infiltration of T-cells and other primary inflammation responses were all inhibited by the administration of anti-HMGB1 antibodies, and the development of artherosclerosis lesions was inhibited by 60%（Fig. 4） . The treatment of the new prophlogistic factor High mobility group box-1（HMGB1）with neutralizing antibodies was applied to mice models of arterial sclerosis, and it splendidly succeeded in inhibiting minor vascular inflammation. Patent Application 3（Artherosclerosis inhibitor, Patent Application 2009-223472） .

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There is, of course, a high possibility that hybrid antibodies will have clinical applications in regards to artherosclerosis, but they may also be effective for treating brain infarctions（brain infarction inhibitor, patent no. 3876325） , cerebrovascular spasms that follow subarachnoid hemorrhages（cerebrovascular spasm inhibitor, Patent no. 3882090） , brain damage and others. It －  133  －

is expected that these antibodies will be used to treat a wide variety of pathological states. Patent application 1 Invention name: Brain infarction inhibitor, Okayama University reference number: OP00134 Basic application: Patent Application 2005-308949（2005/10/24） Status of domestic patent rights: Registered（2006/11/10） , Patent no. 3876325 International application: PCT/JP2006/320436（2006/10/13） International publication: WO2007/049468（2007/05/03） Status of patent family patent rights: EP1946774（A1）2008/7/23 US2009252739（A1）2009/10/08 Details of the invention: Studying inflammation reactions in the brain that occur during the acute phase of cerebral ischemia, it was surmised that it would be possible to reduce the size of the end brain infarction that forms by inhibiting such inflammation. The treatment target chosen was a newly discovered cytokine, the active protein compound known as High mobility group box-1 （HMGB1）. Specific monoclonal antibodies were created to target HMGB1, and antibodies with the ability to neutralize HMGB1 significantly reduced the size of the brain infarction and improved nervous system symptoms in cerebral infarction model rats. Patent application 2 Invention name: Cerebrovascular spasm inhibitor, Okayama University reference number: OP00189 Basic application: Patent Application 2006-140773（2006/05/19） International application: PCT/JP2007/060231（2007/05/18） International publication: WO2007/135992（2007/11/29） Status of domestic patent rights: Registered（2006/11/10） , Patent no. 3882090 Status of patent family patent rights: CN101448524（A）2009/6/3 EP2020241（B） 2010/09/08（Registered） CH2020241（B） 2010/09/08（Registered） DE602007009087.3（B） 2010/09/08（Registered） FR2020241（B） 2010/09/08（Registered） GB2020241（B） 2010/09/08（Registered） US2009175878（A） 2009/7/9 Details of the invention: Studying the inflammation reactions inside the brain during the cerebrovascular spasms that occur frequently following subarachnoid hemorrhages, it was surmised that it is possible to treat the spasms by regulating this inflammation. Attention was given to the new cytokine molecule high mobility group box-1, and specific monoclonal antibodies were created for HMGB1. Antibodies with the ability to neutralize HMGB1 exhibited significant effect in rat models. －  134  －

Patent application 3 Invention name: Artherosclerosis inhibitor, Okayama University reference number: OP00522 Basic application: Patent application 2009-223472（2009/09/28） Status of domestic patent rights: Yet to request an examination International application: PCT/JP2010/066683（2010/09/27） International publication: WO2011/037227（2011/03/31） Status of patent family patent rights: WO2011/037227（2011/03/31） Details of the invention: This invention provides a pharmaceutical that effectively inhibits artherosclerosis and has few side effects. Also, a particular characteristic of this artherosclerosis inhibitor is that it uses anti-HMGB1 monoclonal antibodies that bond to the C tail domain of HMGB1（High mobility group-box 1）as an effective ingredient. Patent application 4 Invention name: Screening method of RAGE and AGE bonding inhibitors Okayama University reference number: OP00675 Basic application: Patent application 2010-214019（2010/09/24） Details of the invention: Please refer to the above for details. Patent application 5 Invention name: Inhibitor of brain damage induced neuropathy, Okayama University reference number: OP00685 Basic application: Patent application 2011-131674（2011/06/13） Details of the invention: Please refer to the above for details. Superiority of the developed technology At the Okayama University Intellectual Property Division, important patents are supported as “Magma patents®.” The above group of patents has been designated as “Magma patents®” and has been deemed to be an important task for the Intellectual Property Division. “Prevention and medication agent related to induction of the functions of（HMGB1）High mobility group box-1 -（AGEs）Advanced glycation end product” and “prevention and medication agent related to the regulation and inhibition of the intranuclear lethality mediator of ⇒ HMGB1 ⇒ AGEs ⇒ RAGE ⇒ Active” are high mobility medications. The target diseases here are related to general lifestyle diseases, but the patent group is particularly related to the prevention and treatment of brain infarctions, brain hemorrhages, arterial sclerosis, dementia, and malignant tumors, and its usefulness is immeasurable. The most important issue in our country’s healthcare is to what extent the various serious diseases that develop from the progression of lifestyle diseases can be prevented. It goes without saying that efforts need to be made to prevent specific diseases such as hypertension, diabetes. As a result, it is possible to prevent brain infarctions and myocardial infarctions, and a great －  135  －

contribution can be made to the solving the healthcare, health economics, and the nursing care/ welfare problem in this country. Also, it is possible that a compound developed during this research can be a RAGE blocker, and in such an event, it will be possible to apply for patent rights for it as a new pharmaceutical.

Schematic view of the Magma patent process as it progresses from a Magma patent to group of several patents to the production of a group of products. Topic Agent for prevention and treatment of lifestyle diseases related to High mobility group box-1 （HMBG1）and its receptor, RAGE（agent for prevention and treatment of diseases related to the control of the intranuclear lethality mediator and its receptor “HMBG1 → RAGE”） “Okayama University Magma Patent® High mobility（medical）” group patent No. G10-40 Subject fields（keywords） ・ Life sciences（medical） ･･ Prevention/treatments related to lifestyle diseases ･･･ Preventive medication for brain infarctions ･･･ Preventive medication for brain hemorrhages ･･･ × Preventive medication for cerebrovascular spasms ･･･ Preventive medication for arterial sclerosis ･･･ Medications for dementia ････ Medications for Alzheimer’s disease ･･･ Medications for malignant tumors ・ High-molecular compounds －  136  －

･･ Antibody ･･･ Anti-HMGB antibody ････ Anti-HMGB monoclonal antibody ・ Low-molecular compound ･･ Inhibitor ･･･ Anti-HMGB inhibitor ･･･ Anti-HMGB receptor（RAGE）inhibitor Contact Person Name

Kenich YOSHIDA

E-mail

[email protected]

Name of department

Intellectual Property Office of Organization For Research Promotion & Collaboration of OKAYAMA University

Position

Manager of Intellectual Property Office

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