Background: Angina (A) in heart disease patients causes disability and reduced Quality of Life (QoL). Patients may still have AP despite revascularization and/or use current anti-angina drugs (AAD). AAD with new mechanism of action would improve AP treatment. T89 is a 3-herb traditional Chinese Medicine completed its US phase 2 and 3 developments ready for New Drug Application to the FDA.

Methods: For FDA registration, pre-IND meetings outlined the entire development track for Dantonic (T89). Full scaled pharmacology/toxicology studies were completed and accepted according to Special Protocol Agreements with the FDA alone with all chemistry, manufacture, and control info. The pivotal randomized, placebo controlled global phase 3 clinical trial in 8 countries and 127 clinical centers was successfully completed and presented to the FDA recently.

Results: T89 acts on several pathological targets including promoting antioxidant, energy metabolism and lipid peroxidation; preventing cholesterol plaque; inhibiting platelet aggregation and adhesion; suppressing thrombus development, and etc. Chronic use of T89 is not characterized by tachyphylaxis or severe toxicities particularly useful in long-term prevention and treatment of A. In vivo Cooperstown 5+1 Cocktail drug-drug interaction (DDI) studies showed no apparent pharmacokinetic DDIs with any of those P450 metabolic substrates or current AAD. T89 did not elevate INR when dosed with warfarin. There was no Peak-Trough effect. The onset of action takes ～2 weeks but was able to maintain at high level without tolerance. Phase 2 and 3 clinical trials show that T89 significantly improves the Total Exercise Duration (TED) compared with placebo in 1,154 patients aged 40-80 yrs with Canadian Cardiovascular Society angina class 2 or 3 on Treadmill. Time to ST Depression, Time to Chest Pain, and QoL were significantly improved (p<0.05) with a time- and dose- response trend. There were >85% reduction of weekly A and Nitrate dose. Adverse effects from all trials were rare, miner and similar in all arms. No QT prolongation was indicative on any treatment days.

Discussion: The successful registration of T89 with FDA will pave a new path for traditional Chinese Medicine to go global and stimulate the worldwide interest in multi-herbal drug discovery and developments.

Although ginseng is the best known herb and has been in human use for a long time, US National Institutes of Health and Food and Drug Administration do not consider ginseng safe of effective. Ginseng's health benefit claim on immune system is discounted as an unapproved claim. Use of ginseng longer than 3 months can be harmful according to US NIH and FDA. Adverse effects cited from ginseng use are insomnia, menstrual problems, breast pain, increased heart rate, negative effects on blood pressure, headache, loss of appetite, diarrhea, itching, rash, dizziness, mood changes, vaginal bleeding, and others. Even such serious uncommeon side effects as Steven-Johnson syndrome and liver damage from ginseng use have been claimed to have taken place. People with diabetes and heart-diseae patients who can benefit from ginseng use are warned not to use ginseng.

For ginseng use to be widely accepted, it is clear to produce scientific and objective evidence that ginseng use is safe and effective. To this end, both interventional and non-interventional studies are needed. In my presentation, I will propose a number of safety and medicinal effect studies. Also I will introduce ginseng safety and efficacy studies in which I have been engaged.

PG2, different from single-ingredient chemically synthetic drugs, is a novel lyophilized injection containing complicated mixture of plant-derived ingredients. PG2's raw materials, Astragalus membranaceus, were cultivated in the specific region of Inner Mongolia, China. Based on the GAP (Good Agricultural Practice) protocols, Astragalus were grown via breeding, nurturing seedlings, field growing, harvesting to monitor soil, water, fertilizer in order to control their qualities and batch-to-batch uniformity. After harvesting, Astragalus underwent step-by-step SOP such as screening, washing, drying, slicing before shipping to Taiwan. Every batch of Astragalus raw material must go through DNA appraisal to confirm it was Astragalus membranaceus by Industrial Technology Research Institute (ITRI), Hsinchu, Taiwan. Astragalus were subsequently manufactured through a series of proprietary processes: extraction, condensation, filtration, purification and spray drying to obtain the highly-purified polysaccharides as the PG2 active pharmaceutical ingredients (API). The entire process from raw material to finished product comprised of 68 steps and 141 inspection items to ensure product quality and uniformity.

Following TFDA's "Guidance for Botanical Drug Clinical Trials" and "Guidance for Botanical Drug New Drug Application", PG2 underwent complete animal toxicology studies and Phases I, II and III human clinical trials before submitting for NDA with the TFDA. This presentation will demonstrate the positive Phase III clinical study outcomes together with the results from an even larger-scale Phase IV post-marketing clinical trials. PG2 was finally approved by the TFDA for treating cancer-related fatigue (CRF), a disease that is seriously associated with chemotherapy and immunotherapy among cancer patients. PG2 has been marketed to more than 30 major medical centers, hospitals, clinics and has treated more than 5,000 late-stage cancer patients in Taiwan.

RANKL was identified as an osteoclast differentiation factor as well as a T cell-derived stimulator of dendritic cells. The essential role of RANKL in osteoclastogenesis has been genetically proven in both mice and humans. In addition, RANKL was shown to play a critical role in various tissues including thymus, gut and mammary gland. Human monoclonal antibody against RANKL, denosumab, has been successfully utilized for the treatment of bone metastasis and osteoporosis. We long studies on the mechanism of bone destruction in rheumatoid arthritis and proposed that RANKL should be an auspicious therapeutic target for arthritis-associated bone destruction. Recently, denosumab has been approved for rheumatoid arthritis in Japan. We have explored the role of RANKL in the immune system and showed its role in the migration of Th17 cells to the central nervous system. We also identified the RANKL-expressing cells in various contexts such as bone remodeling, arthritic bone destruction and gut epithelial M cell development. Here I overview the function of RANKL in bone and the immune system and introduce recent findings in the therapeutic perspective.

During the last decade, intravital optical microscopy has launched a new trend in the field of biology. By using this advanced imaging technique we have established a new system for visualizing in situ behavior of a diversity of living cells within intact tissues and organs. Among them, we succeeded in visualizing the various dynamic phenomena within bones and joints, where various kinds of immune cells are produced and functioning although poorly analyzed by conventional methodology such as histological analyses with decalcified sections. We have so far identified the real modes of migration, differentiation and function of bone-destroying osteoclasts, special kind of macrophages responsible for bone and joint erosions. This novel technique does not only help us to understand the dynamic nature of living cells and tissues in situ, but identifies the in vivo pharmacological actions of several new drugs. Here we show our recent studies for analyzing bone-protecting actions of different biological agents treating RA by directly visualizing drug-induced cellular behaviors in vivo. Such trials would be useful for classifying various biological agents based on their respective pharmacological effects in vivo.

Primordial cartilage serves as the skeletal template during development. It gives rise to two types of cartilage, growth cartilage and articular cartilage, after birth. Growth cartilage is where the bone grows in children, and its dysfunction due to genetic mutations causes short stature and skeletal malformation, conditions called skeletal dysplasia. Articular cartilage covers the ends of bones and provides shock absorption and lubrication to diarthrodial joints. Injury and degeneration of articular cartilage cause joint pain during motion, leading to osteoarthritis in adults. The conditions that compromise growth cartilage or articular cartilage are poorly understood, and curative drugs are not available. iPSC technologies have been used to study cartilage diseases. Cartilage consists of chondrocytes embedded in abundant extracellular matrix that the chondrocytes produce. We have developed a method in which human iPSCs (hiPSCs) are differentiated toward chondrocytes that produce extracelluar matrix to prepare cartilage (hiPSC-derived cartilage).

We are generating effective and safe hiPSC-derived cartilage as regenerative medicine technology to treat damage in articular cartilage and sustain healthy joint function. The goal is to use this cartilage in clinical tests. Currently, we are performing pre-clinical tests by transplanting iPSC-derived cartilage into defects created in the articular cartilage of model animals. We are also analyzing the molecular mechanisms that regulate chondrocyte differentiation and cartilage homeostasis. We have clarified that salt-inducible kinase 3 (SIK3) regulates the differentiation of chondrocytes and maintenance of articular cartilage. SIK3 can be a target molecule for treating articular cartilage damage.

In a separate project, we have generated hiPSC-derived cartilage from patients with skeletal dysplasia. FGFR3 chondrodysplasia such as achondroplasia is caused by a gain-of-function mutation in the FGFR3 gene. We found that chondrocytes derived from hiPSCs generated from patients suffering from FGFR3 chondrodysplasia produce abnormal cartilage that reproduces the pathology of the diseases and thus offers an iPSC-based disease model.

Wnts are secreted glycoproteins and play roles in development and carcinogenesis. Wnt signaling has been subdivided into β-catenin-dependent canonical pathway and several non-canonical pathways such as Ca²⁺ pathway and planar cell polarity (PCP) pathway. From the beginning of this century, studies of rare human diseases have identified that mutations in several components of the canonical Wnt/β-catenin pathway cause bone disorders. Loss-of-function mutations in low density lipoprotein receptor-related protein 5 (LRP5), encoding a Wnt co-receptor, are responsible for osteoporosis-pseudoglioma syndrome (OPPG) characterized by low bone mass. On the other hand, gain-of-function mutations in LRP5 are associated with high-bone-mass trait. Loss-of-function mutations in another Wnt co-receptor LRP6 also lead to low bone mass. Defects in the SOST gene encoding sclerostin, an inhibitor of Wnt signaling secreted by osteocytes, are responsible for sclerosing bone disorders sclerosteosis and van Buchem disease, and inactivation of LRP4, a facilitator of sclerostin action, also causes sclerosteosis. More recently, loss-of-function mutations in Wnt1 have been identified to be a cause of osteogenesis imperfecta and early-onset osteoporosis. Mouse studies using various spontaneous and genetically-engineered mutants confirmed the critical role of canonical Wnt/β-catenin signaling in regulation of bone mass and revealed that activation of Wnt/β-catenin pathway increases bone mass by stimulating osteoblasts bone formation and suppressing bone resorption. Bone anabolic effect of parathyroid hormone (PTH) is partially mediated through the activation of Wnt/β-catenin pathway. In addition to the canonical pathway, accumulating evidence has suggested that non-canonical pathways also play roles in controlling bone mass. Wnt signaling is now a promising therapeutic target for conditions of bone fragility such as osteoporosis and osteogenesis imperfecta. Humanized neutralizing antibodies against sclerostin such as romosozumab (Amgen) and blosozumab (Eli Lilly) have been developed, and preclinical and clinical studies have demonstrated the bone anabolic effects of these drugs. In this presentation, I will review our current understanding on the mechanisms by which Wnt signaling controls bone mass and the clinical application.

In 2016 Pitolisant as first histamine H3 receptor (H3R) inverse agonist/antagonist has received market approval by the European Medicine Agency for the orphan disease narcolepsy with and without cataplexy and is marketed by Bioprojet in whole Europe. Their contract in 2017 with Harmony Bioscience should open the U.S. market for Pitolisant. Several clinical trials on different therapeutic indications with different H3R antagonists have been described. Alzheimer s disease, schizophrenia, multiple sclerosis, alcohol abuse, and obstructive sleep apnea are only a selection of potential indications of clinical trials, mostly without the report on the exact outcome. Actually, the most prominent therapeutic application for Pitolisant and related H3R antagonists could be excessive daytime sleepiness. This somnolence frequently occur with Parkinson patients on dopaminergic medication.

Very recently, we have been able to follow an off-label use of Pitolisant as case study on a small number of children having Prader-Willi syndrome (PWS), a very rare genetic disease with hypotonia, hyperphagia, and cognitive impairments. The preliminary outcome hints on remarkable cognitive and social improvements connected to higher wakefulness and alertness during daytime. For the H3R-related influence on sleep periods during daytime on children with PWS, it is not possible to draw any conclusion so far due to the small and heterogeneous cohort with three patient reports only.

The histamine H₄R is the fourth member of the histamine receptor family. It has a high affinity for histamine and a more restricted expression pattern than some of the other histamine receptors. Histamine, acting via the H₄R on dendritic cells as well as T cells, was shown to be important in the priming, activation and polarization of T cells. Most of the early work was focused on allergic Th2 T cell-driven diseases such as atopic dermatitis and asthma, where conventional wisdom suggested histamine should play a role. However, the effects on T cells prompted an exploration of the role for the H₄R in other T cell mediated diseases. One disease of interest was rheumatoid arthritis. H₄R antagonists were shown to reduce disease in several mouse models of arthritis, thus providing the rationale for testing in clinical studies. Two clinical studies in patients with rheumatoid arthritis were carried out with the selective H₄R antagonist toreforant. Post-hoc analyses in the first study indicated that 100 mg/day of toreforant was associated with reduced rheumatoid arthritis symptoms. However, a larger second study that tested lower doses did not show any benefit relative to placebo. Thus, the role for the H₄R in rheumatoid arthritis is still unclear. One of the preclinical models for rheumatoid arthritis where H₄R antagonists were efficacious is known to be dominated by Th17 T cell responses and indeed the antagonists were able to inhibit Th17 cells in vivo. Therapies that target the Th17 pathway have not been shown to be effective in the treatment of rheumatoid arthritis, but are very efficacious in the treatment of psoriasis. This finding led to a study of toreforant in patients with moderate-to-severe plaque psoriasis. The results showed that toreforant exhibited low levels of efficacy in these patients. The results of the clinical studies with toreforant in rheumatoid arthritis and psoriasis suggest that the H₄R may play a minor role in these diseases, but that the therapeutic benefit of H₄R antagonists in these diseases is not clear.

Allergic rhinitis is a representative incurable disease. Symptoms are not sufficiently improved by current therapeutics. Then the novel therapy having notable alleviation is highly expected. We succeeded in finding the correlation between symptoms and level of histamine H₁ receptor (H1R) mRNA in the nasal mucosa of pollinosis patients. Treatment of antihistamines alleviated allergic symptoms, and suppressed H1R mRNA up-regulation. H1R gene expression was suggested to participate in allergic symptoms.

H1R gene expression was induced by H1R stimulation and protein kinase C-delta (PKC-delta) activation, and H1R up-regulation was not only with antihistamines but also with Kujin, an anti-allergic Kampo medicine. PKC-delta and heat shock protein 90 (HSP90) are known to form complex. (-)Maackiain was successfully identified as an active substance of Kujin, and HSP90 was observed to be a target molecule. Allergic symptoms were alleviated by the treatment of (-)maackiain as well as antihistamines to nasal hypersensitivity model rats.

Combination treatment of antihistamines and suplatast tosilate to the model rats induced notable alleviation of allergic symptoms. Combination treatment of antihistamines and Awa-tea, a local fermented tea in Tokushima, or extract of lotus root showed similar notable alleviation. We discovered that suplatast, Awa-tea and extract of lotus root all suppressed IL-9 gene expression.

In conclusion, H1R and IL-9 gene expressions participate in allergic symptoms, and combination therapy using suppressors of the two gene expressions is expected for notable alleviation of allergic rhinitis. Gene expression of H1R is suggested to be a pathological mechanism of allergic rhinitis, and IL-9 is suggested to be the second one. The intracellular target molecules of (-)maackiain and pyrogallol are expected to be novel drug targets for allergic rhinitis.

Schizophrenia is a highly intractable mental disorder, whose pathogenesis mechanisms remain unclear. Its negative symptoms have been recognized for a long time as the most devastating among all symptoms clusters of schizophrenia without quite effective treatment. The low histamine H1 receptor (H1R) binding has been found in the brains of schizophrenia patients, but the role of H1R in schizophrenia remains controversial and mysterious, suggesting the H1R in different type neurons may play distinct roles in the pathogenesis of schizophrenia. So, we generated mice with selective deletion of H1R in glutamatergic (Hrh1^fl/fl;CaMKIIα^Cre), dopaminergic (Hrh1^fl/fl;DAT^Cre) or cholinergic neurons (Hrh1^fl/fl;ChAT^Cre) to test their schizophrenia-like behaviors. Interestingly, we found that selective deletion of either glutamatergic or dopaminergic has not induced any schizophrenia-like behaviors, even the dysfunction of glutamatergic or dopaminergic system is closely related to the development of schizophrenia. However, selective deletion of H1R in cholinergic neurons (Hrh1^fl/fl;ChAT^Cre) caused schizophrenia-like negative symptoms rather than positive symptoms, including sensorimotor gating deficit in prepulse inhibition measure, anhedonia, abnormal social behavior, which could be rescued by re-expressing H1R in the cholinergic neurons. In addition, Hrh1^fl/fl;ChAT^Cre mice showed dysfunction of cholinergic neurons in basal forebrain, but not in striatum, as well as excitation/inhibition imbalance in medial prefrontal cortex. Pharmacogenetic activation of cholinergic neurons in the basal forebrain rescues schizophrenia-like negative symptoms in Hrh1^fl/fl;ChAT^Cre mice. Moreover, pharmacogenetic inhibition of cholinergic neurons in basal forebrain not only caused schizophrenia-like negative symptoms but also deteriorated the positive symptoms induced by MK-801, which was different from the deletion of H1R in cholinergic neurons. Taken together, our data firstly demonstrated that H1R in cholinergic neurons may be crucial for the development of the negative symptoms of schizophrenia, which could serve as a therapeutic target.

In 1980, Mahidol University (MU) committed to develop a live attenuated tetravalent dengue vaccine. DEN-1, -2 and -4 obtained from DHF patients were serially passaged in certified primary dog kidney (PDK) cells. DEN-3 obtained from DF patients was first passaged in green monkey kidney (GMK) cells and then in certified Fetal Rhesus Lung (FRhL) cells. The degree of attenuation was empirically based on certain biological markers.

The monovalent live attenuated viruses - DEN-1 PDK13, DEN-2 PDK53, DEN-3 PGMK30/FRhL-3 and DEN-4 PDK48 - were first tested in flavivirus non-immune adult subjects, followed by bivalent, trivalent and tetravalent vaccine trial tests. All vaccine recipients developed either a mild or no reaction to the vaccine but elicited satisfactory immunogenicity.

Homotypic neutralizing antibody began to rise 15 days after immunization in vaccine recipients who had no pre-existing antibodies to dengue or Japanese encephalitis (JE) viruses. In the subjects with pre-existing heterologous antibodies against dengue serotypes of JE viruses, the neutralizing antibodies began to rise earlier (7 days after immunization); the peak titers at 1 month post-immunization were many fold higher that the titers seen in non-immune volunteers. The neutralizing antibodies were homotypic in the flavivirus naïve volunteers receiving monovalent vaccines, and the titers remained at about the same level for 3 years with only slight reduction. After 3 years, heterotypic antibodies against the other serotypes began to appear, raising a possibility that the vaccinees might have experienced a subsequent natural infection. For the bivalent and the trivalent formulation, the vaccinees displayed homotypic neutralizing antibodies, with the exception of the bivalent DEN-1-DEN-4 which showed heterotypic antibodies against DEN-2 and DEN-3 as well. These heterotypic antibodies disappeared after 1 year.

None of the small group of vaccinees with pre-existing dengue antibody showed severe reactions to the vaccines. Follow-up physical examination was possible in about half of the vaccinees, some of whom had been immunized nearly 10 years earlier. There were no apparent sequelae in the follow-up group.

From 2013 to 2016, the largest outbreak of Ebola virus disease (EVD) since the disease was first reported in 1976 occurred in West Africa. More than 28,000 cases were reported, and more than 11,000 people died from the disease, primarily in Guinea, Liberia, and Sierra Leone. During this outbreak, we developed the reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for EBOV using a portable device. The validation study in Guinea republic showed that the assay had comparable diagnostic accuracy to that of the reference real-time RT-PCR test for serum samples or oral swabs from suspected EVD cases. In Guinea, a mobile field laboratory with RT-LAMP testing facilities was deployed to improve the laboratory diagnostic abilities in remote areas. The RT-LAMP test can be conducted using a portable and battery-equipped detection system, making it practical for use in the field.

Outbreaks of emerging viral diseases have been reported every year in anywhere of the world. Our assay system could be applied to diagnostics for other viral diseases.

Background

Rabies is a zoonotic lethal encephalitis caused by the rabies virus (RABV), which is usually transmitted by animal bites. The case fatality rate for rabies remains almost 100%, and there is still no means of cure after the onset of symptoms although the development attempt for drugs has been done for many years. Recently, Favipiravir (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has been shown to be active against a broad range of RNA viruses and been expected to be a therapeutic drug for rabies. In this study, we evaluated the antiviral efficacy of T705 against RABV infection in vitro and vivo.

Methods

Antiviral activity against RABV was examined in mouse neuroblastoma cell lines, NA and N2a cells. Cells were inoculated with RABV at an MOI of 0.01 and incubated for 96h in the presence of T705. The viral titers in the media were determined using a focus assay. The antiviral efficacy of T705 in 6-week-old female ddY mice was also evaluated. Mice were intramuscularly inoculated with 105 FFU of the street (wild) strain 1088 and were administered T705 (30, 100, or 300 mg/kg/day, p.o.) or 0.5% methylcellulose (control) daily for 7 or 14 days. The administration was commenced 1h or 4 days post inoculation, or on the day when symptom onset was apparently observed.

Results

A 3-4 log10 reduction in RABV growth in N2a cells treated by T705 (1 mM), but the activity was lower in NA cells than in N2a cells. We detected the lack of HPRT expression which is requisite for conversion T705 to its active form in NA cells and the activity partially restored by the complementation of the expression of hprt gene in NA cells. T705 significantly improved morbidity and mortality in RABV infected mice when orally administered at a dose of 300 mg/kg/day, beginning 1h after inoculation. However, the survival of RABV infected mice was not significantly improved when T705 treatment was initiated 4 days after inoculation or after the onset of symptoms.

Conclusions

Our data suggested that T705 was active against RABV and has the potential to provide an alternative to rabies immunoglobulin in rabies post exposure prophylaxis.

BACKGROUND: Zika virus (ZIKV) is a mosquito-borne flavivirus. ZIKV infection is considered to be the cause of microcephaly in fetuses and newborns and Guillain-Barre syndrome by recent epidemics of ZIKV infection in Brazil. The diagnostic of ZIKV infection is performed in laboratories by nucleic acid amplification tests (rRT-PCR) and serological tests. Since flaviviruses are highly conserved, serologic tests are difficult to use for diagnosis due to cross-reactivity to other flavivirus. The rRT-PCT is mainly used for the diagnosis but requires special techniques and equipment and takes time. Thus, simpler diagnostic tests are desired. Since flavivirus nonstructural protein 1 (NS1) is secreted from virus-infected cells into the bloodstream, this protein is known to remain detectable in the blood even after the viral genome has disappeared and is useful as an infectious disease marker. Therefore, we developed rapid immunochromatographic kits for detection of ZIKV NS1.

METHODS: Monoclonal antibodies (MoAbs) specific to ZIKV NS1 were established by immunizing mice with recombinants or synthetic peptides. Characterizations of the MoAbs were investigated by ELISA using recombinants. Rapid immunochromatographic kits were developed using established MoAbs and then evaluated using recombinants, culture supernatants of ZIKV-infected cells and clinical specimens.

RESULTS: We established three MoAbs (2-42, 1-78 and 84F9) with high reactivity and specificity to ZIKV NS1. Rapid immunochromatographic kits were developed with available three MoAbs. These rapid kits could detect NS1 derived from two ZIKV strains (African and Asian strain) using both recombinants and culture supernatants of ZIKV-infected cell lines. Detection sensitivity of our kits was 125 pg/mL using recombinant NS1. In the testing against five clinical specimens collected in Brazil in 2016, which were confirmed ZIKV infection by rRT-PCR (Ct=19.4-21.6), our kits could detect all specimens as positive and also detect NS1 in over 2000-fold diluted sample within 15 min. Further, the kits showed no cross-reactivity against specimens infected with dengue virus 1 to 4 containing NS1 at high level.

CONCLUSIONS: Our kits constructed in this study could detect NS1 in ZIKV-infected specimens at high sensitivity and specificity and might be useful for clinical application to acute-phase of ZIKV infection.

Eisai is a global research and development based pharmaceutical company committed to improving the health of all individuals worldwide regardless of wealth or geography. We give our first thoughts to patients and their families and to increasing the benefits healthcare provides - a principle that forms the basis of Eisai's human health care (hhc) philosophy, which has been embedded into our Articles of Incorporation and national programs across the globe.

Based upon the concept of hhc philosophy, "Access to Medicines" (ATM) should be improved and secured for all patients. For example, proactive efforts toward developing medicines that address clear needs in developing countries, such as treatments for NTDs and three major infectious diseases, are required to improve ATM, even though pharmaceutical companies in developed countries have not focused on those diseases so far. Eisai is working on various activities in order to deliver its medicines to more patients and people at risk around the world including developing countries without delay.

In 2013 after securing WHO PQ, Eisai initiated the manufacturing and distribution at prize zero to endemic countries worldwide of DEC tablets to patients suffering with Lymphatic Filariasis (LF). In 2017 Eisai committed to supply DEC at prize zero until LF elimination.

Eisai's efforts have produced a rich R&D portfolio of assets addressing the needs of four Tropical Diseases: Malaria, TB, Chagas Disease and LF through collaborations with Academic partners, PDPs and Governmental Organizations.

A novel model was developed in order to provide sustainability to Global Health R&D activities. We have successfully secured funding for programs through grants obtained from external organizations such as the Global Health Innovative Technologies Fund (GHIT, Japan), Bill & Melinda Gates Foundation (BMGF, US), US Department of Defense, US NIH.

A brief list of ongoing programs developed through the strategy described include: the development of E1224 (originally an antifungal) for Chagas disease and Mycetoma; the development of SJ733 (an ATP4ase inhibitor), the identification and selection of a novel Phe tRNA ligase inhibitor (a novel target), and a GWT1 inhibitor (a novel target) as a novel antimalarial, respectively. Details of some programs will be presented.

We have developed a new gene amplification method, loop-mediated isothermal amplification (LAMP), which has the potential advantages in rapidity, simplicity, and specificity. Because of the aforementioned characteristics, LAMP is recognized as one of ideal gene amplification methods that can be applied in resource-limited facilities in developing countries.

Tuberculosis (TB) is one of the greatest threats to global public health, infecting approximately one-third of the world's population. Sputum smear tests are widely used for the detection of TB. However, they do not offer sufficient specificity and sensitivity for screening TB patients effectively. While the culture methods provide the most sensitive and reliable TB detection and have used as the standard of TB diagnostics, it has a disadvantage in rapidity since it often takes 4-6 weeks to obtain the results. Simple, rapid and sensitive TB diagnostic technology has been desired for effective TB control and elimination.

In order to meet the above demand, we have developed a new TB diagnostic method named TB-LAMP. One of the key technologies composing the TB-LAMP is the sample processing method named PURE (Procedure for Ultra Rapid Extraction) that can remove inhibitory components from unprocessed sputum samples in approximately 10 minutes. The other important technology is the formulation of LAMP reagents. Formulation in dried form enables us to transport and store the LAMP reagents at room temperature without relying on the expensive infrastructures such as cold chains and freezers.

The recommendation from World Health Organization (WHO) is essential for introducing a technology into the most developing countries. In order to obtain the recommendation to TB-LAMP, we conducted the evaluation study of TB-LAMP in various countries and successfully earned the recommendation in 2016. Because of its simplicity, rapidity and robustness, TB-LAMP is now widely recognized as a WHO recommended genetic test and begins to be implemented in some developing countries.

The LAMP technologies are also being applied to the diagnostics of malaria and some neglected tropical diseases. We hope to contribute to the improvement of global public health and to the realization of universal health coverage through the development of the effective diagnostics using LAMP.

A game-changing mechanism of developing new treatments for neglected patients has been awaited because the current capitalism in private sector seldom allows to develop new treatments without profit. One of the mechanisms to overcome this stereotype is so-called Product Development Partnership (PDP) which enables to develop new treatments through collaboration of pharmaceutical companies, universities, research institutions and CROs with financial support from funding agencies.

Recently Global PDPs have been actively performing R&D on drugs, vaccines or diagnostics etc. with public and private funding. On this platform PDP often plays as a project manager of the virtual R&D team which consists of expertise from collaborating partners. This mechanism can offer better treatments to patients with diseases that fall outside the scope of market-driven R&D.

In 1999, Médecins Sans Frontières (MSF) brought together a team of international experts to study the crisis in drug R&D for neglected diseases. One of the reasons why MSF decided to create this Drugs for Neglected Diseases working group was that they found many patients suffering from tropical diseases and lack of efficient treatments because most of patients live in low-income countries. DNDi was officially established in 2003 as a global PDP with 7 founding partners and has put a great amount of effort to achieve R&D of inexpensive, efficacious, field-adapted treatments to address neglected patients' needs.

One of the greatest achievements is Fexinidazole, a treatment for African Human Trypanosomiasis, so-called sleeping sickness, which was found as a hit compound in DNDi's global screening on Nitroimidazoles and is about to be approved by EMA after a long way of development processes. Another achievement is the Neglected Tropical Diseases Drug Discovery Booster as a platform of screening and Hit to Lead process, through which millions of chemical compounds in partners' libraries can be tested in a speedy and cost- effective way.

In humans, the brain develops over a long period of time, from the early embryonic days to the postnatal year. Cellular and molecular mechanisms on the brain development have been studied mostly in model animals and post-mortem specimens. However, it is uncertain whether such studies fully reveal the mechanisms that occur in living humans. Emergence of human induced pluripotent stem cells (iPSCs) provides us an opportunity to investigate the development and dysfunction of the human brain. We previously developed 3D culture systems using human PSCs that recapitulate in vivo human brain development. We utilized the self-organizing principles that autonomously appear in the cell population of human PSC reaggregates after neural differentiation. We succeeded in generation of 3D cerebellar tissues that correspond to the cerebellar anlage of human embryos. 3D reaggregate culture followed by long-term 2D co-culture with other neural progenitors enabled to differentiate cerebellar neuronal precursors into mature cerebellar neurons, including Purkinje cells with elaborated dendritic arbors. These culture techniques were further applied to investigation of neurological disorders. We have succeeded in generation of differentiated Purkinje cells from spinocerebellar ataxia (SCA) patient-derived iPSCs by combining the 3D self-organizing culture and the 2D co-culture for maturation. We demonstrated that SCA-derived Purkinje cells exhibit vulnerability to severe culture environment and that this vulnerability is suppressed by treatment with existing drugs. The platform based on the iPSC technology and the self-organizing stem cell 3D culture will become a powerful tool for investigating human brain development and the neuronal diseases.

ES and iPS cells belong to pluripotent stem cells that have an ability to differentiate into various cells and undergo sustained growth in vitro. Many studies demonstrated that ES/iPS cells mimic the disease phenotypes in vitro and are powerful tools not only for understanding the molecular mechanisms underlying the pathogenic events but also for developing the new drugs in the future.

Disease-derived iPS cells are generated from somatic cells by over-expressing four reprogramming factors including Oct-3/4, Sox2, Klf4 and cMyc. In particular, the iPS cells are quite useful for studying the intractable diseases as the number of the biopsy samples is limited.

We have generated various kinds of intractable disease-derived iPS cells from the patients' skin fibroblasts and blood cells by Sendai virus (SeV) vector. We concentrated into the study for iPS cells from patients with inherited metabolic diseases such as Niemann-Pick disease type C (NPC). The lipid metabolism was markedly impaired in both the NPC-derived hepatocyte-like and neural cells, resulting in the cholesterol accumulation in both cells. The treatment with Cyclodextrin and its derivatives greatly reduced this accumulation in the patient-derived cells and restored the cellular functions. We are also studying other inherited metabolic diseases. In this symposium, we will present our recent works using the disease-derived iPS cells.

Organogenesis generates higher-order structures containing functional subunits, connective components, and progenitor niches. Despite recent advances in organoid-based modeling of tissue development, recapitulating these complex configurations from pluripotent stem cells (PSCs) has remained challenging. The kidney derives from the embryonic metanephros, which develops by the reciprocal interactions between the nephron progenitor and ureteric bud. We previously identified the distinct origins of these two precursor tissues and generated the nephron progenitor from mouse and human PSCs (Taguchi et al. Cell Stem Cell 2014). The induced nephron progenitors readily formed glomeruli and renal tubules in vitro, and upon transplantation, human glomeruli were vascularized with the host mouse endothelial cells (Sharmin et al. J Am Soc Nephrol 2016). We also developed a protocol to expand the nephron progenitors in vitro while retaining their nephron-forming potential (Tanigawa et al. Cell Rep 2016).

　More recently, we reported an induction protocol for the ureteric bud from mouse and human PSCs, by studying the developmental processes of this second lineage, which contains epithelial kidney progenitors that undergo branching morphogenesis and thereby plays a central role in orchestrating organ geometry. Importantly, mouse organoids reassembled from the differentially induced ureteric bud and nephron progenitors developed the inherent architectures of the embryonic kidney, including the peripheral progenitor niche and internally differentiated nephrons that were interconnected by a ramified ureteric epithelium (Taguchi et al. Cell Stem Cell 2017). This selective induction and reassembly strategy will be a powerful approach to recapitulate organotypic architecture in PSC-derived organoids.

Cancer arises through the accumulations of both genetic and epigenetic alterations. Although the causal role of genetic mutations on cancer development has been established in vivo, similar evidence for epigenetic alterations is still limited. Moreover, mutual interactions between genetic mutations and epigenetic alterations remain unclear. Cellular reprogramming technology can be used to actively modify the epigenome without affecting the genomic information. Here I introduce our recent studies that utilized this property for cancer research.

　Cell type-specificity of cancer development has long been recognized. However, the underlying mechanism how particular genetic mutations transform a specific cell type remains unclear. Clear cell sarcoma (CCS), which is a rare soft tissue sarcoma caused by EWS/ATF1 fusion gene, resembles malignant melanoma, and thus is known as "malignant melanoma of soft parts". Here, we established induced pluripotent stem cells (iPSCs) from EWS/ATF1-controllable murine CCS cells harboring sarcoma-associated genetic abnormalities. Sarcoma-iPSC mice developed secondary sarcomas immediately after EWS/ATF1 induction, but the sarcomas arose exclusively in the soft tissue. Taking advantage of the one-step, cell type-specific sarcoma model, we identified a cell-of-origin for these sarcomas. Furthermore, we showed that EWS/ATF1 is preferentially recruited at enhancer of the cell-of-origin in CCS cells. All together, cancer cell reprogramming uncovered cell-of-origin for CCSs and provided a molecular basis for cell type specificity of cancer development.

The Drug Toxicity Signature Generation Center (DtoxS.org), is a NIH funded center to obtain cellular signatures for toxicity associated with therapeutic agents. Adverse events involving diminished cardiac contractility leading to heart failure is associated with efficacious protein kinase inhibitors used to treat various cancers. My colleagues and I in this center have sought to develop clinically-weighted transcriptomic signatures to predict risk of kinase inhibitor drug induced cardiotoxicity and to better understand the biological processes associated with risk of cardiotoxicity. We obtained transcriptome-wide response profiles in human primary cardiomyocyte like cell lines and healthy human subject iPSC derived myocytes that were treated with twenty two different FDA approved kinase inhibitor drugs. The FDA Adverse Event Reporting System was used to derive risk scores for these drugs for different types of cardiotoxicity. We used elastic net regression to associate these transcriptomic profiles with kinase inhibitor-associated risk scores for cardiac disease subtypes to obtain clinically-weighted transcriptomic signatures, which showed good predictive properties: the signature could predict relative cardiotoxicity risk for two kinase inhibitors not included in the analysis. The gene products in the signatures form closely connected protein-protein interaction networks. Multidimensional analyses indicate structural features of these drugs could also be selectively associated transcriptomic signatures and clinical risk. These studies indicate that transcriptomic signatures can be useful early predictors of adverse events during drug development.

We remember the crucial role Darrell Abernethy played in promoting the science of predictive toxicology and dedicate these studies to his memory.

Drug-induced liver injury (DILI) remains a burden to the public, the pharmaceutical industry and regulators. Not only do we generally lack clear mechanisms that can explain the injury, but the manifestations of DILI are diverse, they are difficult to diagnose, and also they can take weeks or even months to develop. Whilst there are few good animal models of DILI, there are similarly few good in vitro models at present. Therefore, we believe that it is important that we understand what our in vitro/in vivo models are fit for as well as their limitations. Otherwise, model development will not be a rational scientific exercise. This talk will explore the challenging area of liver toxicity and stem cell models of hepatotoxicity, from the perspective of the work being done in the MRC Centre for Drug Safety Science, from our role as coordinator of the IMI MIP-DILI project, and will review recent advances in this area made by other groups. The following key themes and questions will be addressed in this talk: The importance of phenotyping developing stem cell models, based on iPS and tissue-derived stem cells, using not only transcriptomic, but also proteomic and functional assays; What does hepatotoxicity look like in an animal model, and can we emulate this in a dish? How should new stem cell-based technologies be used appropriately in hepatotoxicity safety assessment? Can biomarkers help to relate data derived in vivo and in humans back to in vitro models?

Abstract not yet available.

Oceania comprises the large islands of Australia, New Zealand and Papua New Guinea (PNG) and a very large number of islands comprising Melanesia, Micronesia and Polynesia. There are few pharmacogenomic studies and results cannot be readily predicted from one region to another. For example, CYP2C19 genotypic poor metabolisers (*2,*3) comprise almost 70% of the Melanesians in Vanuatu, less than 10% in Maori populations and less than 5% in Aboriginal Australians, and in the latter population, the gain of function *17 results in 17% rapid metabolisers, half that of Caucasians. Such large differences are in contrast to CYP2D6 poor metabolisers (*4,*5) which comprise less than 1% in most of the regions studied.

In PNG, most studies have focused on infectious diseases pharmacogenomics. The frequency of CYP2B6*6 is 60% compared to 20% in Caucasian and Asian populations. Efavirenz is metabolized by CYP2B6 and poor metabolizer status is associated with CNS/Psychiatric effects; in 52 subjects, only drowsiness was related to *6 carrier status. For CYP2D6, the *5 frequency was 5% and *1/N duplication 12%, both having implications for p.vivax treatment with primaquine. Minimal data are available on drug transporters in Oceania, however in PNG subjects the frequency of the C3435T variant was 67%, with almost 90% of subjects being either homozygous variant or heterozygotes. The implications of this finding for the efficacy and adverse effects to many medicines, especially those used for infections diseases, is noteworthy. For NAT2 and acetylator status, no genomic studies have been conducted in PNG but almost all are rapid acetylators, and therefore the incidence of isoniazid-induced hepatotoxicity is rare. The phenytoin HLA B*13:01 is associated with severe hypersensitivity reactions prevalent in several Asian countries and with a frequency of almost 25% in PNG and in Aboriginal Australians from Northern Australia. The implications for precision medicine in Oceania are several. Although the frequency of some important pharmacogenes are markedly different to Caucasian and Asian populations, the frequencies are not widespread across the region; many important genes, and genotype-phenotype correlations, have not been assessed and clinical relevance and translation need to be viewed in relation to limited resources.

Sub Saharan Africans represent the most genetically diverse population in the world, characterized by extensive population substructure and less linkage disequilibrium between loci. Pharmacogenetic research in Africa is progressing through regional and international research collaborations. The African pharmacogenomics consortium is established to prompt research and deliver guidelines for the use of pharmacogenomics-based tests in African populations.

Africa is affected by a high prevalence of HIV/AIDS, tuberculosis, and malaria. Co-infection of these diseases is common, but co-treatment is challenging due to drug interactions and overlapping toxicities. Pharmacogenetic studies focusing on the treatment optimization of HIV/AIDS, and its comorbidities are conducted in different African populations. However, between-country extrapolation of data is not feasible due to vast population genetic diversity. CYP2B6 metabolizes antiretrovirals such as efavirenz and nevirapine as well as antimalarial drugs. Defective CYP2B6 variant alleles occur at a higher frequency in black-African ancestries. The frequency of CYP2B6*6 allele, which is associated with high efavirenz plasma exposure, drug induced liver and neuropsychiatric toxicities, reach up to 40%. Genome-wide association studies revealed potential novel genetic biomarkers for antiretroviral and anti-tuberculosis drugs induced liver toxicities. HLA-B*57 alleles (B*57:03 and B*57:02) confer susceptibility to antituberculosis and antiretroviral drugs induced liver toxicity. HLA-C*04:01 allele is associated with nevirapine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis in sub-Saharan Africans.

The importance of pharmacogenetic variations in modulating interactions between antiretroviral, antituberculosis, antimalarial drugs is noted. Reduced efavirenz metabolism in black Africans, partly due to CYP2B6 genotype counterbalancing the effect of CYP enzyme induction by rifampicin, necessitated different efavirenz dosing strategy during rifampicin-efvairenz co-treatment. CYP2B6 genotype based efavirenz dose modification for sub-Saharan Africa population is recently recommended. Bed-side pharmacogenetics test for genotype-based drug dosing, its clinical use and applicability remain to be investigated to translate pharmacogenetic research into clinical practice in Africa. In this presentation, key pharmacogenetic studies in different African populations, with focus on treatment optimization of HIV/AIDS and its comorbidities such as tuberculosis and malaria will be presented. Effect of pharmacogenetic variations on first-line antiretroviral, antituberculosis and antimalarial drug interactions, and its impact on treatment outcomes will be summarized.

Pharmacogenetics/pharmacogenomics relies on human genetic diversity. My talk will initially focus on the pharmacogenomic consequences of human demographic history and genetic diversity. I will then move to the more formal pharmacogenetic arena and discuss the impact of population diversity on the worldwide distribution of common variants in pharmacogenes, using the NAT2 gene as an example. Inference of NAT2 phenotypes across global populations by a tag SNP or combinations of 2 - 4 SNPs will be contrasted with the standard 7-SNP panel. Next, the pharmacogenomic implications of population admixture will be examined, drawing data from the two most populous countries in Latin America, i.e. Brazil and Mexico, where a kaleidoscopic combination of individual proportions of Native American, European and sub-Saharan African ancestries prevails. Accordingly, average admixture proportions are not predictive of the corresponding proportions at the individual level. Thus, ethnic/race/Color categories cannot capture the complex structure of Latin American peoples. As a consequence of admixture, the genetic differentiation of common pharmacogenetic variants among Brazilians is much attenuated in comparison to their relevant ancestral populations, especially Europeans and Africans. The impact of such attenuation on the predictive performance of warfarin dosing algorithms in Brazilians, compared to their ancestral proxies, will be discussed. Studies in admixed populations have the potential to reveal novel pharmacogenomic associations: a distinct example is the disclosure of gene-dose effect of CYP3A5 polymorphisms on the dose requirement of tacrolimus, first reported in Brazilian renal transplant patients. Pharmacogenomic implementation in clinical practice must take into account the abundance of rare variants (minor allele frequency <1% or <0.5%) in our genome. Due to their recent origin there is very little sharing of rare variants between continental populations or within continental populations. A substantial effort will be required to catalogue rare variants and to develop reliable algorithms to identify their putative functional effects and potential clinical relevance.

Pharmacogenomic researches have been performed in high levels also in Asia, especially in association analyses with severe cutaneous adverse reactions. Association of an HLA type, HLA-B*15:02 with carbamazepine-related Stevens-Johnson syndrome and toxic epidermal necrolysis was first reported from Taiwanese and its association is specific to Asian populations since HLA-B*15:02 is frequent in South-East Asians and Chinese. In addition, HLA-B*58:01, HLA-B*51:01, HLA-A*02:06, HLA-A*02:07 and CYP2C9*3 were associated with severe cutaneous adverse reactions related to allopurinol, phenobarbital, cold medicine such as acetaminophen, zonisamide and phenytoin, respectively, which were first reported from Asian researchers. Other examples of functional genetic polymorphisms in relatively high frequencies for Asian populations are UGT1A1*6 with reduced enzymatic activities and CYP2C19*2 and *3 with null activities. UGT1A1 is a metabolic enzyme for active metabolite of irinotecan, SN-38, and UGT1A1*6 in addition to UGT1A1*28 are associated with severe neutropenia. CYP2C19 is involved in the metabolism of omeprazole and the patients with the null allele (*2 and/or *3) show higher success rates of Helicobacter pylori eradication. Helicobacter pylori infection is one of the major factors for gastric cancer in East Asians.

On the other hand, their implementation is still limited. In Taiwan, prior testing of HLA-B*15:02 for the carbamazepine-administred patients should be considered in patients with ancestry in Asian populations and the test is reimbursed by the national health insurance. But in Japan, only the description of its association is on the package inserts, probably due to the low allele frequency in Japanese. UGT1A1*6 and *28 testing is reimbursed by national health insurance, but the test is not mandatory.

Issues on collaboration among Asian countries are 1) differences in used drugs between developing countries and advanced countries, 2) poverty of cold chain in sampling and storage processes in some countries, 3) difficulty in exporting genomic DNA to the other countries at least in a few countries/regions.

In this presentation, major achievements of Asian pharmacogenomic researchers and their implementation are summarized.

The hypothalamus has a critical role in the regulation of energy and glucose homeostasis. Over-nutrition triggers profound cellular and physiological changes of the hypothalamus diminishing hypothalamic responses to insulin and leptin, critical hormones maintaining normal blood glucose and body weight. This decline in hypothalamic responses has been proposed as crucial processes that underlie the pathophysiology of metabolic disease, and its mechanism is of particular interest in understanding the neural basis of overnutrition-associated diseases. Through a combination of ex vivo brain explant assays, electrophysiology, genetic, and pharmacologic studies in mice, we recently identified EPAC (the GTP/GDP exchange factor directly activated by cAMP), and its effector, the small GTPase Rap1, as a molecular pathway that responds to nutrient excess, promotes hypothalamic pathological changes and mediates obesity. Because the Epac-Rap1 pathway can be activated by G protein-coupled receptors (GPCRs) via G protein Gs-cAMP signaling, we searched for a GPCR ligand that activates EPAC-RAP1 signaling and might also cause hypothalamic hormone resistance. Of the candidate GPCR ligands, we identified the gut hormone gastric inhibitory polypeptide (GIP), whose levels are elevated during diet-induced obesity, as a factor that caused hypothalamic resistance to leptin and insulin in ex vivo brain explants. Moreover, centrally administered GIP in vivo caused obesity-associated hypothalamic pathogenic events via its receptor GIPR. Global gene expression profiling analysis revealed that brain signaling pathways most affected by GIP are inflammatory-related; inflammation is known to induce leptin and insulin resistance. Our data suggest that GIP administration is sufficient to recapitulate key aspects of hypothalamic pathophysiology of obesity. More importantly, blocking brain GIPR action with a monoclonal antibody reversed body weight and adiposity effects and normalized the impaired hypothalamic metabolic regulatory pathways of dietary obese mice. Collectively, our studies reveal GIP acts as a signal that arises from excess caloric intake and drives hypothalamic pathophysiological changes, and also unveil a previously unrecognized neural pathway connecting GIP/GIPR signaling to hypothalamic pathology in obesity, via neuronal EPAC-RAP1 signaling.

G-protein-coupled receptors (GPCRs) have traditionally been regarded as receptors for hormones, neurotransmitters, and other mediators which are produced solely for the purpose of carrying a signal and to serve cell-cell communication. This view has changed during the last decade, as a growing number of GPCRs are being identified, for which the ligands are energy substrates or metabolic intermediates. Among these ligands are saturated and unsaturated free fatty acids (FFAs) as well as hydroxy carboxylic acids (HCAs), such as lactate and ketone bodies, which exert cellular effects through GPCRs named FFA1-FFA4 and HCA1-HCA3, respectively. These receptors are widely expressed in the human body and regulate the metabolic, endocrine or immune system to maintain homeostasis under changing dietary conditions. Data will be presented on the role of FFA1, FFA2 and FFA3 in the regulation of beta-cell function and of the lactate receptor HCA1 in adipocyte function and body weight regulation by natural ligands acting in part in an autocrine manner.

Long-chain fatty acids (LCFA) are important regulators of pancreatic islet function. Acutely, LCFA amplify glucose-induced insulin secretion from pancreatic beta cells. However, when present at excessive levels for prolonged periods of time, LCFAs impair beta-cell function. The discovery of G protein-coupled receptors (GPCRs) that are activated by LCFA in the early 2000s has prompted major drug discovery efforts to target these receptors for the treatment of metabolic diseases. Of these, FFA1 (GPR40) is predominantly expressed in pancreatic beta cells. While some controversy remains as to whether prolonged FFA1 activation would have undesirable side effects, most drug discovery programs have focused on the development of FFA1 agonists to enhance insulin secretion in type 2 diabetes. Studies from our group have provided evidence that FFA1 contributes to the maintenance of glucose homeostasis under normal conditions and in response to insulin resistance in rodents. However, clinical development of an FFA1 agonist was abruptly halted due to liver toxicity. FFA4 (GPR120) is structurally divergent but shares the same endogenous ligands with FFA1. The expression pattern of FFA4 is more ubiquitous, and its activation of this receptor in adipocytes and macrophages has been proposed to mediate the anti-inflammatory and insulin-sensitizing effects of omega-3 fatty acids, although this remains controversial. In islets, FFA4 is preferentially expressed in somatostatin-secreting delta cells and its activation inhibits somatostatin secretion, which indirectly stimulates insulin secretion. Therefore, FFA1 and FFA4 appear to play complementary roles in pancreatic islet function. However, many aspects of the biology and pharmacology of these receptors remain to be clarified.

Abstract not yet available.

The first gastroprotective drugs (e.g., sofalcone, sucralfate) which prevent and/or accelerate healing of gastric ulcers, without inhibiting acid secretion, were first introduced in Japan, before Andre Robert's historic article on "gastric cytoprotection" in 1979. Since Robert's studies were focused on prostaglandins (PG), they became the center of GI research for more than 30 years. Endogenous PG were implicated in mediating the gastroprotective effect of other drugs (e.g., sofalcone, sucralfate), despite that the COX inhibitor indomethacin diminished, but never abolished gastroprotection by other drugs. Another endogenous substance, i.e., sulfhydryls (SH), investigated in parallel with PG, also play a mechanistic role in gastroprotection, esp., since SH alkylators like N-ethylmaleimide counteract virtually any form of gastroprotection.

In Robert's terms as 'prevention of chemically induced acute mucosal lesions', no single mechanism could explain the beneficial effects of diverse protective agents, but I argue that two endogenous substances (i.e., PG, SH), in addition to histamine, are the main mechanistic mediators of acute gastroprotection: PG & histamine, because as mediators of acute inflammation, they increase vascular permeability (VP), & SH scavenge free radicals. Based on research work of the last about 35 years, in part from our lab, a new mechanistic explanation of gastroprotection may be formulated: it's a complex but orderly & evolution-based physiologic response of the gastric mucosa under pathologic conditions. Namely, one of the first physiologic defense responses of any organ is inflammation that starts with rapid vascular changes [e.g., increased VP and blood flow], followed by cellular events (e.g., infiltration by acute and chronic inflammatory cells). Thus, PG and histamine, by increasing VP create a perivascular edema that dilutes and delays toxic agents reaching the subepithelial capillaries. Increased mucus and/or bicarbonate secretion causes luminal dilution of gastrotoxic chemicals that is further reinforced by a perivascular, histodilutional component. This mechanistic explanation would encompass the protective actions of diverse agents such as PG, small doses of histamine, motility stimulants, dilute irritants (i.e., "adaptive cytoprotection"). Thus, although markedly increased VP is pathologic, slight increase in VP seems to be protective, i.e., a key element in the complex pathophysiologic response during acute gastroprotection.

Kampo, a distinctively unique Japan's traditional herbal medicine, is fully integrated into the modern health care system in Japan, it is neither a folk remedy nor alternative therapy in Japan. Kampo medicines are dispensed at all the university, national, and foundation hospitals as prescription drugs, frequently in combination with western drugs. Rooted in Chinese medicine, Kampo followed a decidedly unique path of development in Japan.

Since 1986 daikenchuto (DKT) has been prescribed for the treatment of two symptoms: abdominal bloating and cold sensation in the abdomen and Japanese government insurance started to cover the medical fee of DKT. DKT is the most frequently prescribed as Kampo medicine in Japan, especially in the field of gastroenterology. The formulation of DKT is composed of extract granules of Japanese pepper, processed ginger, and ginseng radix with maltose powder. There has been a recent surge in scientifically robust data from basic and clinical studies for DKT. Clinical studies on DKT, including placebo-controlled double-blind studies for various gastrointestinal disorders, and pharmacokinetic studies, have been conducted or are in the process of being conducted in both Japan and the USA. Clinical studies suggest that DKT is beneficial for postoperative complications, especially ileus and abdominal bloating. Pharmacokinetic and basic studies indicate that the effect of DKT is a composite of numerous actions mediated by multiple compounds supplied via multiple routes. In addition to known mechanisms of action via enteric/sensory nerve stimulation, novel mechanisms via the TRPA1 channel and two pore domain potassium channels have recently been elucidated. DKT has also effects on improving intestinal blood flow. The critical players responsible for vasodilatory effects are the two peptides, calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM). Another important factors for understanding the mechanism of action of DKT is through study of the receptors involved. When DKT stimulates CGRP and ADM, simultaneously up-regulates CRLR and RAMPs, and develops up-regulation of CGRP and ADM receptors. DKT compounds target these channels with and without absorption, both before and after metabolic activation by enteric flora, with different timings and possibly with synergism. We advocate the worldwide availability of Kampo medicines.

Background: Gastrointestinal prokinetic agent, serotonin-4 receptor (5-HT₄R) agonist, mosapride citrate (MOS) activates myenteric plexus neurons to release acetylcholine (ACh), which in turn induces anti-inflammatory action, but detailed pathway remains to be elucidated. Aim: Aim of this study is to clarify the anti-inflammatory mechanism of intramural plexus stimulation via 5-HT₄R on leukocytes infiltration in animal model for postoperative ileus (POI). Methods: Postoperative ileus was induced by intestinal manipulation in wild type C57BL6/J (WT), 5-HT₄R knock-out (S4R KO), α7nAChR knock-out (α7R KO) and M2 muscarinic AChR (M2AChR) knock-out (M2R KO) mice. Results: Intestinal manipulation induced intestinal muscularis inflammation with leukocytes infiltration, inflammatory cytokines upregulation, and motility disorder. Intramural plexus stimulation via 5-HT4R attenuated leukocytes infiltration in WT. The intramural plexus stimulation-induced anti-inflammatory action by MOS was completely suppressed in S4R KO mice, and also in chimera SR4 KO mice, which had wild type bone marrow-derived immune cells and SR4 KO body cells. On the other hand, the intramural plexus stimulation-induced anti-inflammatory action against macrophages infiltration but not neutrophils infiltration was attenuated in α7R KO mice. Selective α7nAChR agonists, PNU-282987 and AR-R17779 also inhibited only macrophages infiltration. Furthermore, α7nAChR could be detected in intestinal resident macrophages by immunohistochemistry using anti-FLAG Tag antibody in FLAG-tagged α7nAChR knocked-in mice. In contrast, the intramural plexus-mediated inhibition of neutrophils infiltration was diminished by atropine, M2AChR antagonist, methoctramine and in M2R KO mice. Conclusion: 5-HT₄R stimulation inhibits leukocytes infiltration in POI possibly through intramural plexus activation. Released ACh inhibited macrophages and neutrophils infiltrations by activation of α7nAChR presumably on macrophages, and M2AChR, respectively. Thus, macrophages and neutrophils recruitment into the inflamed site is inhibited by different types of AChRs in small intestine.

Specific brain-gut peptides have been identified to modulate vagal activity regulating visceral function. Namely, medullary thyrotropin-releasing hormone (TRH) acting on TRH1 receptor in the dorsal motor nucleus of the vagus (DMN) plays a physiological stimulatory role. In particular, we showed that the stable TRH analog, RX77368 activates DMN neurons and gastric vagal efferent discharges, myenteric cholinergic neurons and upregulates gastric peripheral choline acetyl transferase (pChAT) expression leading to a rapid increase gastric blood flow, secretion, motility and gut hormone release. Ghrelin is a gastric hormone that also activates gastric vagal efferent activity. There is recent evidence that the vagus exerts cholinergic anti-inflammatory action as shown by electrical vagal stimulation and that intestinal inflammation is a relevant mechanism in postoperative ileus. We recently investigated whether abdominal surgery (AB) induces also gastric inflammation that can be modulated by brain-gut peptides. At 6-h after AB, gastric emptying (GE) was inhibited by 72% vs control. Inflammation was detected in whole mount preparation of gastric myenteric plexus (MP) where AB doubled the number of M1-like macrophage immunoreactive for MHCII (M1 marker) but not for CD206 (M2 marker) (MHCII+/CD206-) while there was no change in M2-like macrophages (MHCII-/CD206+). AS also increased mRNA levels of interleukin-1 (IL-1) beta and tumor necrosis factor (TNF) alpha in the gastric submucosa plus muscle layers by 1.7- and 1.5-fold respectively and the infiltration of neutrophils labeled by myeloperoxidase in the muscularis externa by 9.5-fold. There was a significant negative correlation between GE and gastric IL-1beta, TNF-alpha, M1 macrophage and neutrophils. RX77368 injected intracisternally 1-h before AB inhibited these AS-related gastric inflammatory changes and dampened GE while not altering these parameters in sham group. Likewise, the orally active, brain penetrant ghrelin agonist, HM01 activates neurons (Fos number) in the MP and DMN and hexamethonium reduced MP activation by 2 fold while not influencing that in DMN. Oral HM01 pretreatment abolished AS-induced delayed GE and increased IL-1beta and TNF-alpha expression while elevating IL-10 in both sham and AS groups. These data showed that central vagal activation by TRH or ghrelin may provide new venues to counteract AB-induced gastric inflammation and correlated gastric ileus.

The edict “No Acid, No Ulcer” (Schwarz Beit Clin Chir 1910, led to efforts in the twentieth century to inhibit acid secretion. The most successful early intervention was vagotomy (Dragsted et al PSEBM 1943) which was then superseded by H2 antagonists (Black et al Nature 1972). These show uniform tolerance and short duration of action making them relatively ineffective but still were a major breakthrough in treatment. They were then replaced by PPIs that are acid activated covalent inhibitors of the gastric H,K ATPase (Fellenius et al Nature 1981) that show long duration of action and no tolerance. Omeprazole binds to cys813 and cys892 in the proton pathway (Besancon et al JBC 1997). However, the need for acid activation and a short plasma half-life restricts their efficacy even at bid dosing. The first potassium competitive inhibitor (PCAB) of the gastric H,K ATPase, SCH28080, had liver toxicity and was an imidazopyridine with a short plasma half-life and rapid dissociation (Wallmark et al JBC 1987) and many other imidazopyridine were tested and dropped. Recently a pyrrolopyridine (vonoprazan Hori et al JPET 2011) is on the market and others are in development with relatively long half-lives and immediate onset of action that likely will supersede PPIs. This compound's N-methylmethaneamine (pKa9.2) binds at glu343 blocking K binding and ATPase activity (Abe et al Nature 2018). It is now clear that infection by Helicobacter pylori is responsible for most peptic ulcer diseases where 1% of infected individuals succumb to the disease a rate 10 times higher than non-infected people and therefore eradication of the infection could be a new modality of treatment. Antibiotic resistance has made eradication difficult and generally unpredictable without measurement of antibiotic resistance. However, it appears that lengthy acid inhibition (pH >5.0 24hrs per day) stimulates bacterial division and hence amoxicillin sensitivity (Marcus et al APT 2012) and amoxicillin resistance is rare compared to resistance to other antibiotics. However, H. pylori expresses beta-lactamase so resistance may appear. Elevation of the organism's periplasmic pH is essential for acid survival and urease and periplasmic carbonic anhydrase are essential for this to occur. The elevation of periplasmic pH has been shown directly by measurement of eGFP fluorescence (Wen et al J Bact 20180 Expression of this carbonic anhydrase remains high at pH 3.0 in contrast to urease (Marcus et al Helicobacter 2018) suggesting its importance for gastric survival. An attractive eradication strategy would be then to use a long lasting carbonic anhydrase inhibitor with twice a day dosing to provide an antibiotic free means of eradication and this inhibitor is already available in the form of Diamox sequels. Treatment with Diamox resulted in a recurrence rate of 6% similar to that found after eradication of H. pylori compared to 43% following conventional medical therapy and with hindsight this is likely due to eradication of the infection (Puscas ANYAS 1984). It has indeed been shown that its periplasmic carbonic anhydrase is important for gastric survival of H. pylori and is required for neutralization of periplasmic pH in acid (Marcus et al J Bact 2005). So, today, the dictum might become “No Hp, no ulcer”.

For the past three decades, proton-pump inhibitors (PPIs) have been the mainstay of treatment of acid related disorders including gastroesophageal reflux disease and nonsteroidal anti-inflammatory drug (NSAID)-induced peptic ulcers. There is good evidence that PPIs heals upper gastrointestinal mucosal ulcerations through profound acid suppression. Nevertheless, certain high risk patients still develop recurrent ulcers or ulcer bleeding despite receiving regular PPIs, suggesting that acid suppression with PPIs is either incomplete or other factors contribute to recurrent mucosal damage. Recent evidence suggests that combination of PPI and a COX-2 selective NSAID offers the best upper GI protection in arthritis patients with high GI and high cardiovascular risks. Whether vonoprazan, a potassium-competitive acid blocker translates into superior clinical outcome remains uncertain. Recently, there are concerns about the negative impact of PPI on gut dysbiosis and other possible systematic side effects.

Many patients are taking a variety of medicines for the treatments of different kinds of disorders. PKs and PDs of some of medicines are often influenced by concomitant medicines, such as gastric acid inhibitors. Gastric acid inhibitors are often used with non-steroidal anti-inflammatory drugs (NSAIDs) and antiplatelet agents, such as low dose of aspirin and clopidogrel for the prevention of upper gastrointestinal bleeding. Patients with symptoms of heartburn and/or dyspepsia are sometimes treated with a gastric acid inhibitor. However, absorption of some drugs are decreased or increased by gastric acid inhibition. For examples, it is well known that gastric acid inhibitors inhibit the absorption of ketoconazole and that Cmax and bioavailability of ketoconazole is decreased. Gastric acid inhibitors also influence the absorption of some of molecular target agents.

Further more, some of acid inhibitors, such as proton pump inhibitor (PPI) and cimetidine, influence the hepatic metabolism of some medicines. It has been long discussed whether a PPI attenuates the antiplatelet function of clopidogrel to increase the risk of cardiovascular events of patients treated with clopidogrel. Recently, a new type of gastric acid inhibitor, vonoprazan, categorized to the potassium competitive acid blocker (P-CAB) has been clinically available. The gastric acid inhibition attained by vonoprazan is superior to PPIs, such as esomeprazole and rabeprazole. The eradication rates of H. pylori and cure rates of erosive esophagitis by vonoprazan-based regimens are superior to those of PPIs. However, we found that vonoprazan more potently attenuated the antiplatelet function of clopidogrel than esomeprazole. Interestingly, the influence of vonoprazan on the prasugrel appeared weaker than that on clopidogrel. Optimal selection of acid inhibitor should be performed by not only the need of the acid inhibitory effect but also the risk of drug-drug interaction with other medicines.

Proton pump inhibitors (PPIs) was developed from thiocarboxamides derivatives in 70s. Currently, various PPIs are similar in chemical structure and thus are highly effective and relatively safe. PPIs are used in 60% of inpatients with little benefit in half, resulting in long-term use as outpatients. Recent clinical data have indicated various potential problems associated with long-term use of PPIs, including pneumonia, Clostridium difficile infection, bone fracture, malabsorption, cognitive impairment and dementia, fundic gland polys, hyperplasia of ECL cells and gastric carcinoids (ECLoma) and even gastric cancer. Possible underlying mechanisms involve PPI-induced hypochlorhydria, hypergastrinemia, PPI per se and its metabolites. Animal experimental data showed that the ECL cell hyperplasia reflected the transiently elevated ECL cell proliferation rate during the early phase but was not associated with an accelerated rate of mitosis during the late phase of PPI treatment (52 weeks), suggesting that there are additional factors than hypergastrinemia that involve in the tumorigenesis of ECLoma. In addition to histamine and pancreastatin, the ECL cells produce a blood calcium-lowing peptide which involve in bone mineralization. Long-term use of PPI was associated with an impaired function of the ECL cells as indicated by formations of large vacuoles and lipofuscin bodies and absence of secretory vesicles in the cytoplasm. Thus, hypochlorhydria-induced bone loss may be partly due to the so-called "ECL-cell disease". Hypergastrinemia acts on CCK-2 receptor to stimulate the gastric stem cells and to induce apoptosis in gastric epithelial cells, which might contribute to the development of gastric cancer in mice. Thus, we recommend to limit use of PPIs to those patients who benefit most and to develop new class of drugs targeting the CCK-2 receptor.

Transplantation is the treatment of choice for patients with end-stage renal, cardiac, liver and lung disease. The remarkable success of solid organ transplantation can in large part be attributed to the development of potent immunosuppressive therapy which has reduced the incidence of acute allograft rejection and allograft loss.

In kidney transplantation, tacrolimus-based immunosuppression is the standard in most centers worldwide. Most kidney transplant recipients are treated with induction therapy (consisting of T lymphocyte-depleting antibodies or an interleukin-2 receptor blocker) followed by maintenance therapy consisting of tacrolimus combined with mycophenolate and glucocorticoids.

The (nephro)toxicity of tacrolimus has prompted extensive research into alternative immunosuppressive strategies. Many of these alternative immunosuppressive regimens however, have failed to replace tacrolimus-based immunosuppression because of either unacceptably higher acute rejection rates or because of unanticipated toxicity.

Two immunosuppressive therapies have shown promise as realistic alternatives for tacrolimus-mycophenolate combination maintenance therapy. The first of these is belatacept-based immunosuppression. Belatacept is a co-stimulatory signal blocking agent that is not nephrotoxic. Long-term follow-up data has demonstrated that belatacept-based immunosuppression improves kidney allograft and patient survival as compared with ciclosporin-based immunosuppression. However, the increased acute rejection rates seen during belatacept treatment, the comparison with ciclosporin rather than tacrolimus in the registration studies, and manufacturing problems may limit widespread adoption of belatacept as the primary immunosuppressant. The second alternative is everolimus plus low-exposure calcineurin inhibitor (either tacrolimus or ciclosporin) combination therapy. In the TRANSFORM study, the largest trial ever performed in kidney transplantation with n = 2037 patients, this regimen was compared with standard-exposure calcineurin inhibitor plus mycophenolate therapy. In TRANSFORM, low-exposure calcineurin inhibitor plus everolimus therapy was shown to be non-inferior in terms of the primary end-point consisting of biopsy-proven acute rejection or an eGFR <50 ml/min per 1.73 m2. Cytomegalovirus and BK virus infections were less frequent in the everolimus arm than in the mycophenolate arm.

It is imagined that the benefits of these two alternative immunosuppressive regimens will be further explored in the next decade. In addition, novel compounds, specifically targeting B lymphocyte responses will likely be developed and tested clinically.

A mammalian target of rapamycin (mTOR) inhibitor everolimus (EVR) is approved as one of the immunosuppressive drugs for the prevention of graft organ rejection as well as the agent against various types of cancer and tuberous sclerosis complex. EVR in combination with cyclosporine was approved as an immunosuppressive regimen for the patients after heart transplantation in 2007 in Japan. After that, EVR was also approved for the prevention of graft kidney rejection in 20111. Tacrolimus has been used as a primary calcineurin inhibitor (CNI) in patients after kidney transplantation, and therefore, it is important to establish the methodology of EVR monitoring including the appropriate target range.

Minimizing the use of CNI after organ transplantations reduces long-term complications, including nephrotoxicity and myelosuppression2,3. It was reported that another mTOR inhibitor sirolimus reduced interstitial fibrosis and glomerular sclerosis after kidney transplantation in patients with chronic allograft nephropathy4. In the subtotal nephrectozised (5/6Nx) rats, which are well acknowledged as a good animal model of chronic renal failure in human, marked activation of the mTOR pathway was found at glomeruli and proximal tubules in remnant kidneys5. EVR treatment reduced the expression levels of the phosphorylated S6 which is a marker of activated mTOR. In addition, EVR-treatment restored the tubular reabsorption of albumin, suggesting the regeneration from the functional impairment of tubular epithelial cells5.

Taken together, both of the results of the clinical trials and animal experiments suggest that EVR in combination with the minimized CNI would be a strong strategy in patients after organ transplantation preventing long-term CNI-induced nephrotoxicity such as fibrotic renal disease.

References:

1 Highlights of prescribing information of ZORTRESS® (everolimus) (2016).

2 van Gelder, T., Fischer, L., Shihab, F. & Shipkova, M. Transplant Rev 31, 151-157 (2017).

3 Shipkova, M. et al. Ther Drug Monit 38, 143-169 (2016).

4 Chan, L. et al. Transplantation 85, 821-826 (2008).

5 Nakagawa, S., Masuda, S., Nishihara, K. & Inui, K. Biochem Pharmacol 79, 67-76 (2010).

Grant information: KAKENHI from MEXT Japan (15H04666)

Pharmacometric analysis has assisted clinicians and pharmaceutical scientists in defining dosing regimen of immunosuppressive drugs in heterogeneous transplant populations. It is an important quantitative methodology that can identify and quantify influential covariates to pharmacokinetics (PK) and pharmacodynamics (PD). In particular, population PK models of drugs have been used as a basis of individualized dosing which is also known as "precision dosing". This represents the predefined covariate-based dosing algorithm and therapeutic drug monitoring (TDM)-based dose adjustment through Bayesian adaptive control methodology in transplant patients.

　Age-dependent changes in physiological parameters, e.g. organ sizes and hematocrit, are components which influence the PK/PD of immunosuppressant drugs in pediatric transplant patients. These should be considered in addition to influential covariates identified in adult studies such as time after transplantation, co-medications, liver and kidney functions and genotypes. However, it is always challenging to identify the contribution of these type of covariates in pediatric studies which tend to have a relatively small number of highly variable patients and sparse sample sizes.

　My presentation will provide a comprehensive overview of recent findings on age-dependent changes in the PK/PD of immunosuppressive drugs (tacrolimus, mycophenolate mofetil, biologics etc.) in pediatric transplant populations. This includes our a priori model-guided assessment of reported covariates identified in pediatric population PK/PD modeling and the potential utility of integrative physiologically-based PK/PD modeling techniques to enhance the mechanistic understanding of the drugs. It will also address the potential application of pharmacodynamic markers of immunosuppressive drugs in clinical practice.

The serious side effects and complications related to the use of immunosuppressants in organ transplantation have fueled research into their possible minimization. Immunosuppressive therapy in organ transplantation is therefore tentatively moving from a phase of empirical administration towards individualized therapy. This process is highly dependent on the development of monitoring methods to detect individual immune states.

　Anti-donor alloreactivity, which depends on the number and phenotype of alloreactive precursors in the recipient, could be used to monitor the immune state for individualizing immunosuppressive therapy. For this purpose, we have applied the mixed lymphocyte reaction (MLR) assay, an assay in which CFSE-labeled PBMCs from recipients are cultured with allogeneic lymphocytes from donors as stimuli, to clinical liver and kidney transplantation. This method allows for the separate quantification of the proliferation of CD4 and CD8 responder T cells in response to allo-stimulation by using multiparameter FCM. By analyzing the proliferation and CD25 expression for the CD4 and CD8 T-cell subsets in response to anti-donor and anti-third party stimuli, the immune status was categorized as hypo-, normo-, or hyperresponsive. In patients with hyperresponsive immune status on either CD4 or CD8 T cells, immunosuppressants were increased. In patients with normoresponsive immune status, immunosuppressant tapering was abandoned. Only in patients with hyporesponsive immune status, immunosuppressant therapy was tapered off.

　In addition to cellular ex vivo assays such as the MLR, analyses by molecular genetics such as the single nucleotide polymorphism (SNP) testing would lead to an improvement in risk prediction, early detection and prevention of various complications after organ transplantation. We have recently demonstrated that Fc-gamma R SNPs are predisposing factors for bloodstream infections and can predict mortality after living donor liver transplantation. We have also investigated the impact of SNPs in the FOXP3 gene, a master regulator gene of regulatory T cells, on rejection severity in liver transplant recipients. We found that FOXP3 SNP rs3761548 A/C could be a predisposing factor for steroid-resistant acute rejection after liver transplantation.

　In this session, I would like to introduce how we practice individualizing immunosuppressive therapy by making use of cellular immunology and molecular genetics in clinical organ transplantation.

Physiologically-based pharmacokinetic (PBPK) models include drug-specific and physiology-specific components. This feature allows simultaneous evaluation of the effects of multiple patient factors on pharmacokinetics and pharmacodynamics of a drug in humans. The last decade observed exponential increase in the use of PBPK to address drug development questions. Today, global regulatory bodies are routinely reviewing PBPK analyses submitted by drug sponsors. Among the US FDA, EMA and Japan's PMDA, discussions are ongoing with regard to recommendations on determining adequacy of a PBPK analysis. In 2016, FDA and EMA published first draft guidance and guideline on format and content of PBPK analyses submitted for review. Confidence of using PBPK varies depending on the predictive performance for an intended use. Predictive performance is generally established in the area of predicting enzyme based drug-drug interaction potential for investigational drug as victims. Under certain conditions, PBPK-simulated interaction magnitude is considered adequate by regulators to replace a dedicated clinical interaction trial. Simulated results have been included in product labels in the US, EU, and Japan. Confidence in using PBPK to predict drug PK in other areas are emerging. It continues to be challenging to predict PK in a specific population (e.g., pediatrics, patients with renal or hepatic impairment) using PBPK.

There are several reasons why drug research in children remains challenging, in spite of legal and regulatory initiatives providing incentives to pharmaceutical companies applying for market authorization. The population designated as 'children' is not a homogeneous group, but consists of a range of ages and weights varying from premature neonates weighing less than 500 grams to 17-year old adolescents with a body weight over 100 kg. Especially in the younger age groups sufficient information concerning the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs is lacking. This may lead to under- or overdosing. No generally applicable formula exists to calculate the optimal dose or dose interval for various age groups, since the development of the organ systems responsible for absorption, distribution, metabolism and excretion is not linearly related to age or weight. In addition, the sensitivity of the end organs, determining pharmacodynamics (PD) can vary during development, which may lead to unexpected, sometimes adverse effects.

Before conducting a randomised clinical trial (RCT) to evaluate the efficacy of a drug in children, it is important not only to determine the adequate sample size, but also to determine the optimal dosage. To limit the patient burden of these PKPD studies, the number of subjects, the number of samples per subject and the amount of blood per sample should be minimized, and the timing of sampling should be flexible. The emergence of new laboratory techniques and statistical tools such as NONMEM® allows for the collection and analysis of sparse and unbalanced data, enabling the implementation of (observational) PKPD studies in the paediatric clinic.

The PharmaCool study is an example of a study in which the population PK of drugs frequently used in term neonates with hypoxic ischemic encephalopathy due to perinatal asphyxia undergoing moderate hypothermia (i.e. antibiotics, antiepileptics, analgesics, and sedatives) is described. In this multi-center observational cohort study performed in the Netherlands and Belgium, 12 neonatal intensive care units collaborated to obtain valid data to optimize the dosing of these drugs in this specific patient population.

PBPK models are often used to perform inter- or intra-species scaling, to explore the underlying mechanisms responsible for observed PK characteristics and to simulate receptor occupancy or target suppression in tissues for PD analysis. The PK of macromolecular drugs is driven by mechanisms differing from their small molecule counterparts (Ferl et al. 2016). For monoclonal antibodies (MAbs), transendothelial processes (extravasation and endocytic uptake) and target kinetics are significant drivers of distribution and plasma clearance (Malik et al. 2017), whereas these processes are mostly unimportant for describing the PK of small molecules.

In the era of personalized medicine, we aim to use large molecule PBPK models to explore the PK variability associated with current dosing strategies and to advance our understanding of PK in special populations, such as children.

Rather than considering only the mean individual, virtual populations with real-world variability in physiologic characteristics can be used as inputs for population-level PBPK simulations. The output describes not only the mean PK profile, but also the resulting PK variability for the population as a function of the underlying physiologic characteristics of its individuals. Sensitivity analyses allow us to identify which of these characteristics are most influential to variability in each component of the PK profile. Where these characteristics are clinically identifiable, individualized dosing strategies can be implemented to improve safety and efficacy. Already a PBPK model has uncovered the time and disease-state dependency of PK variability for one MAb and hypotheses have been generated to link the key physiologic determinants of variability to clinically-identifiable characteristics (Malik et al. 2017).

Of interest is the scaling of MAb PK from adults to children for the planning of pediatric clinical trials. A comprehensive review of the age-dependence of the important drivers of large molecule PK and PK variability in children has been completed and will be discussed.

Ferl G, Theil FP, Wong H. 2016. Biopharmaceutics & Drug Disposition. 37: 75-92.

Malik PRV, Hamadeh A, Phipps C, Edginton AN. 2017. Journal of Pharmacokinetics and Pharmacodynamics. 44(3): 277-290.

Physiologically-based pharmacokinetic (PBPK) modelling has been increasingly used within the pharmaceutical industry and also gained acceptance in regulatory agencies over the past years. This technology makes use of prior physiological information that is relevant for the understanding and description of absorption, distribution, metabolization and elimination of the organism of interest as well as physico- and biochemical data of the drug candidate of interest.

Throughout the different stages of drug development, PBPK modelling can be applied in a descriptive manner for hypothesis testing, which physiological processes are most probably leading to observed animal or human PK data, or in a predictive manner to translate PK knowledge for example from animals to humans, from adults to children, or from healthy subjects to patients with certain pathophysiological conditions.

In this talk, recent application examples of PBPK modelling and simulation including potential benefits but also limitations of this technology will be discussed from the view point of the pharmaceutical industry.

SNCA multiplication is a cause for familial Parkinson's disease (PD). We have identified several families with SNCA multiplication. Molecular characterization was performed using real-time PCR, SNP arrays, and haplotype analysis. We further studied those patients who were found to harbor SNCA duplication with olfactory function tests, polysomnography, and PET. We identified six families and one sporadic patient with SNCA duplication, and one family with SNCA triplication, respectively. The lifetime estimate of overall penetrance was ca. 40%. FDG-PET study of symptomatic patients showed hypometabolism in the occipital lobe, whereas asymptomatic carriers of SNCA duplication demonstrated normal glucose metabolism. Symptomatic patients showed abnormal olfactory function and polysomnography and asymptomatic carriers showed normal results. The clinical features of SNCA duplication include parkinsonism with or without dementia. Asymptomatic carriers displayed normal test results with the eldest individual aged 79 years; thus, even a carrier of SNCA duplication may escape the development of PD. This difference in age-associated penetrance may be due to the genetic background or environmental exposures. Further studies of SNCA multiplication carriers will help identify disease-modifiers and may open novel avenues for future treatment. IN addition to SNCA multiplication, we have identified homozygous A53V mutation. This is the first homozygous missense mutation. Clinical features revealed mild phenotypes even though the presence two allele mutations. In this symposium, I will discuss the relationship between clinical phenotypes and genotypes including gene dosage.