Applications of model simulation in pharmacological fields and the problems of theoretical reliability.

The use of mathematical models has become increasingly prevalent in pharmacological fields, particularly in drug development processes. These models are instrumental in tasks such as designing clinical trials and assessing factors like efficacy, toxicity, and clinical practice. Various types of models have been developed and documented. Nevertheless, emphasizing the reliability of parameter values is crucial, as they play a pivotal role in shaping the behavior of the system. In some instances, parameter values reported previously are treated as fixed values, which can lead to convergence towards values that deviate substantially from those found in actual biological systems. This is especially true when parameter values are determined through fitting to limited observations. To mitigate this risk, the reuse of parameter values from previous reports should be approached with a critical evaluation of their validity. Currently, there is a proposal for a simultaneous search for plausible values for all parameters using comprehensive search algorithms in both pharmacokinetic and pharmacodynamic or systems pharmacological models. Implementing these methodologies can help address issues related to parameter determination. Furthermore, integrating these approaches with methods developed in the field of machine-learning field has the potential to enhance the reliability of parameter values and the resulting model outputs.

[The potential of RANKL reverse signaling as a novel pharmacological target].

RANKL is a bidirectional signaling molecule; activation of the RANKL reverse signaling is triggered by the cross-linking of multiple RANKL trimers to form a molecular cluster. In adults, RANKL is expressed primarily in bone tissue and the immune system. In bone tissue, the functions of RANKL forward and reverse signaling have been separately analyzed; the forward signaling is responsible for bone resorption by inducing the maturation of osteoclasts, while the reverse signaling is activated by vesicular RANK, one of the osteoclast-derived coupling factors, leading to the promotion of early osteoblast differentiation. In the immune system, RANKL is expressed on lymphocytes and the interaction with antigen-presenting cells such as RANK-expressing dendritic cells should result in the activation of both the forward and the reverse signaling, however, the discrimination of the function of each pathway has not been achieved yet. To activate RANKL reverse signaling, a multivalent protein construct of anti-RANKL single-chain Fv multimerized with peptide linkers would be effective, since this type of construct can induce cluster formation by cross-linking RANKL trimers. It was also found that the divalent construct with minimal molecular size can cross-link the RANKL trimer without affecting the forward signaling. On the other hand, the design of constructs to inhibit the activation of RANKL reverse signaling needs to be tested experimentally. Particularly, it may be necessary to obtain small molecules that act on the RANKL intracellular domain to achieve selective inhibition of the reverse signaling without affecting the forward signaling.

Utility of constraints reflecting system stability on analyses for biological models.

Simulating complex biological models consisting of multiple ordinary differential equations can aid in the prediction of the pharmacological/biological responses; however, they are often hampered by the availability of reliable kinetic parameters. In the present study, we aimed to discover the properties of behaviors without determining an optimal combination of kinetic parameter values (parameter set). The key idea was to collect as many parameter sets as possible. Given that many systems are biologically stable and resilient (BSR), we focused on the dynamics around the steady state and formulated objective functions for BSR by partial linear approximation of the focused region. Using the objective functions and modified global cluster Newton method, we developed an algorithm for a thorough exploration of the allowable parameter space for biological systems (TEAPS). We first applied TEAPS to the NF-κB signaling model. This system shows a damped oscillation after stimulation and seems to fit the BSR constraint. By applying TEAPS, we found several directions in parameter space which stringently determines the BSR property. In such directions, the experimentally fitted parameter values were included in the range of the obtained parameter sets. The arachidonic acid metabolic pathway model was used as a model related to pharmacological responses. The pharmacological effects of nonsteroidal anti-inflammatory drugs were simulated using the parameter sets obtained by TEAPS. The structural properties of the system were partly extracted by analyzing the distribution of the obtained parameter sets. In addition, the simulations showed inter-drug differences in prostacyclin to thromboxane A2 ratio such that aspirin treatment tends to increase the ratio, while rofecoxib treatment tends to decrease it. These trends are comparable to the clinical observations. These results on real biological models suggest that the parameter sets satisfying the BSR condition can help in finding biologically plausible parameter sets and understanding the properties of biological systems.

Study on Horizon Scanning by Citation Network Analysis and Text Mining: A Focus on Drug Development Related to T Cell Immune Response.

Certain innovative technologies applied to medical product development require novel evaluation approaches and/or regulations. Horizon scanning for such technologies will help regulators prepare, allowing earlier access to the product for patients and an improved benefit/risk ratio. This study investigates whether citation network analysis and text mining of scientific papers could be a tool for horizon scanning in the field of immunology, which has developed over a long period, and attempts to grasp the latest research trends. As the result of the analysis, the academic landscape of the immunology field was identified by classifying 90,450 papers (obtained from PubMED) containing the keyword "immune* and t lymph*" into 38 clusters. The clustering was indicative of the research landscape of the immunology field. To confirm this, immune checkpoint inhibitors were used as a retrospective test topic of therapeutics with new mechanisms of action. Retrospective clustering around immune checkpoint inhibitors was found, supporting this approach. The analysis of the research trends over the last 3 to 5 years in this field revealed several candidate topics, including ARID1A gene mutation, CD300e, and tissue resident memory T cells, which shows notable progress and should be monitored for future possible product development. Our results have demonstrated the possibility that citation network analysis and text mining of scientific papers can be a useful objective tool for horizon scanning of life science fields such as immunology.

Study on Horizon Scanning with a Focus on the Development of AI-Based Medical Products: Citation Network Analysis.

Horizon scanning for innovative technologies that might be applied to medical products and requires new assessment approaches to prepare regulators, allowing earlier access to the product for patients and an improved benefit/risk ratio. The purpose of this study is to confirm that citation network analysis and text mining for bibliographic information analysis can be used for horizon scanning of the rapidly developing field of AI-based medical technologies and extract the latest research trend information from the field. We classified 119,553 publications obtained from SCI constructed with the keywords "conventional," "machine-learning," or "deep-learning" and grouped them into 36 clusters, which demonstrated the academic landscape of AI applications. We also confirmed that one or two close clusters included the key articles on AI-based medical image analysis, suggesting that clusters specific to the technology were appropriately formed. Significant research progress could be detected as a quick increase in constituent papers and the number of citations of hub papers in the cluster. Then we tracked recent research trends by re-analyzing "young" clusters based on the average publication year of the constituent papers of each cluster. The latest topics in AI-based medical technologies include electrocardiograms and electroencephalograms (ECG/EEG), human activity recognition, natural language processing of clinical records, and drug discovery. We could detect rapid increase in research activity of AI-based ECG/EEG a few years prior to the issuance of the draft guidance by US-FDA. Our study showed that a citation network analysis and text mining of scientific papers can be a useful objective tool for horizon scanning of rapidly developing AI-based medical technologies.

Mechanisms of RANKL delivery to the osteoclast precursor cell surface.

RANKL is biosynthesized as a single-pass transmembrane protein, and soluble molecular species are produced by enzymatic cleavage at the cell surface. Recent studies have revealed that the transmembrane form of RANKL is a major contributor to the induction of mature osteoclasts under physiological conditions in vivo. In osteoblasts and osteocytes, most newly synthesized RANKL forms a protein complex with OPG and is selectively sorted to lysosomes. Only the small proportion of newly synthesized RANKL that does not form a complex with OPG is transported to the cell surface. Then, the transmembrane RANKL is delivered to the surface of osteoclast precursors to stimulate RANK, and induces the activation of a downstream signaling pathway. The ability of osteocytes to support the formation of mature osteoclasts appears to depend upon the amount of RANKL molecules present on their cell surfaces. However, the way in which osteocytes, which are embedded in the bone matrix, deliver transmembrane RANKL to the cell surfaces of osteoclast precursors, which are localized in the bone marrow cavity, remains to be elucidated. Further studies are needed to clarify the mechanisms underlying this process.

RANKL as a key figure in bridging between the bone and immune system: Its physiological functions and potential as a pharmacological target.

RANKL is a key molecule that bridges the bone and immune systems. RANKL stimulation activates a signaling pathway downstream of RANK, thereby determining the extent of bone resorption by inducing osteoclast maturation. The signaling pathway also regulates the development of different lymphoid organs, including the thymus, lymph nodes, and Peyer's patches, and plays an essential role in the establishment of immune tolerance. Such characteristics have continued to attract the attention of many researchers, even though it is now more than 20 years since RANKL was identified as a novel member of the TNF superfamily. Recently, we found that RANKL can function not only as a signal input molecule but also as a signal receptor to activate the RANKL reverse signaling pathway, which mediates the coupling between bone resorption and formation. This new finding may provide an important basis for elucidating the complex physiological roles played by RANKL.

Integrative and theoretical research on the architecture of a biological system and its disorder.

An organism stems from assemblies of a variety of cells and proteins. This complex system serves as a unit, and it exhibits highly sophisticated functions in response to exogenous stimuli that change over time. The complete sequencing of the entire human genome has allowed researchers to address the enigmas of life and disease at the gene- or molecular-based level. The consequence of such studies is the rapid accumulation of a multitude of data at multiple levels, ranging from molecules to the whole body, that has necessitated the development of entirely new concepts, tools, and methodologies to analyze and integrate these data. This necessity has given birth to systems biology, an advanced theoretical and practical research framework that has totally changed the directions of not only basic life science but also medicine. During the symposium of the 95th Annual Meeting of The Physiological Society of Japan 2018, five researchers reported on their respective studies on systems biology. The topics included reactions of drugs, ion-transport architecture in an epithelial system, multi-omics in renal disease, cardiac electrophysiological systems, and a software platform for computer simulation. In this review article these authors have summarized recent achievements in the field and discuss next-generation studies on health and disease.

A Novel Method to Estimate Long-Term Chronological Changes From Fragmented Observations in Disease Progression.

Clinical observations of patients with chronic diseases are often restricted in terms of duration. Therefore, obtaining a quantitative and comprehensive understanding of the chronology of chronic diseases is challenging, because of the inability to precisely estimate the patient's disease stage at the time point of observation. We developed a novel method to reconstitute long-term disease progression from temporally fragmented data by extending the nonlinear mixed-effects model to incorporate the estimation of "disease time" of each subject. Application of this method to sporadic Alzheimer's disease successfully depicted disease progression over 20 years. The covariate analysis revealed earlier onset of amyloid-ß accumulation in male and female apolipoprotein E ε4 homozygotes, whereas disease progression was remarkably slower in female ε3 homozygotes compared with female ε4 carriers and males. Simulation of a clinical trial suggests patient recruitment using the information of precise disease time of each patient will decrease the sample size required for clinical trials.

The induction of RANKL molecule clustering could stimulate early osteoblast differentiation.

We recently found that the membrane-bound receptor activator of NF-κB ligand (RANKL) on osteoblasts works as a receptor to stimulate osteoblast differentiation, however, the reason why the RANKL-binding molecules stimulate osteoblast differentiation has not been well clarified. Since the induction of cell-surface receptor clustering is known to lead to cell activation, we hypothesized that the induction of membrane-RANKL clustering on osteoblasts might stimulate osteoblast differentiation. Immunoblotting showed that the amount of RANKL on the membrane was increased by the RANKL-binding peptide OP3-4, but not by osteoprotegerin (OPG), the other RANKL-binding molecule, in Gfp-Rankl-transfected ST2 cells. Observation under a high-speed atomic force microscope (HS-AFM) revealed that RANKL molecules have the ability to form clusters. The induction of membrane-RANKL-OPG-Fc complex clustering by the addition of IgM in Gfp-Rankl-transfected ST2 cells could enhance the expression of early markers of osteoblast differentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results suggest that the clustering-formation of membrane-RANKL on osteoblasts could stimulate early osteoblast differentiation.

Coupling of bone resorption and formation by RANKL reverse signalling.

Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis.

Peptide drugs accelerate BMP-2-induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling.

Both W9 and OP3-4 were known to bind the receptor activator of NF-κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide-induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3-4 accelerated BMP-2-induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL-binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP-2-induced bone regeneration by the RANKL-binding peptides.

Membrane depolarization regulates intracellular RANKL transport in non-excitable osteoblasts.

Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (VD3) are important factors in Ca(2+) homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca(2+) level ([Ca(2+)]i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD3 increased the membrane-bound RANKL (mbRANKL) level at 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca(2+)]i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers for L-type voltage-gated Ca(2+) channels and Ca(2+) release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD3-induced RANKL-lv fusion included the membrane-bound VD3 receptor (1,25D3-MARRS receptor). Elucidating the mechanism of RANKLiT regulation by PTH and VD3 will be useful for the development of drugs to prevent bone loss in osteoporosis and other bone diseases.

Elucidation of the molecular mechanisms underlying adverse reactions associated with a kinase inhibitor using systems toxicology.

BACKGROUND/OBJECTIVES: Targeted kinase inhibitors are an important class of agents in anticancer therapeutics, but their limited tolerability hampers their clinical performance. Identification of the molecular mechanisms underlying the development of adverse reactions will be helpful in establishing a rational method for the management of clinically adverse reactions. Here, we selected sunitinib as a model and demonstrated that the molecular mechanisms underlying the adverse reactions associated with kinase inhibitors can efficiently be identified using a systems toxicological approach. METHODS: First, toxicological target candidates were short-listed by comparing the human kinase occupancy profiles of sunitinib and sorafenib, and the molecular mechanisms underlying adverse reactions were predicted by sequential simulations using publicly available mathematical models. Next, to evaluate the probability of these predictions, a clinical observation study was conducted in six patients treated with sunitinib. Finally, mouse experiments were performed for detailed confirmation of the hypothesized molecular mechanisms and to evaluate the efficacy of a proposed countermeasure against adverse reactions to sunitinib. RESULTS: In silico simulations indicated the possibility that sunitinib-mediated off-target inhibition of phosphorylase kinase leads to the generation of oxidative stress in various tissues. Clinical observations of patients and mouse experiments confirmed the validity of this prediction. The simulation further suggested that concomitant use of an antioxidant may prevent sunitinib-mediated adverse reactions, which was confirmed in mouse experiments. CONCLUSIONS: A systems toxicological approach successfully predicted the molecular mechanisms underlying clinically adverse reactions associated with sunitinib and was used to plan a rational method for the management of these adverse reactions.

Establishment of optimized in vitro assay methods for evaluating osteocyte functions.

Recent studies have revealed that osteocytes play multiple important physiological roles. To analyze osteocyte functions in detail, an in vitro experimental system for primary osteocytes would be useful. Unfortunately, osteocytes tend to dedifferentiate and acquire osteoblast-like features even when the cells are cultured in three-dimensional (3D) collagen gel. Therefore, it is desirable to establish osteocyte culture conditions that prevent dedifferentiation over longer periods. In this study, we obtained systematic information about the influence of culture conditions on osteocyte differentiation states. Fetal bovine serum (FBS) concentrations from 0.1 to 0.5 % in 3D culture matrix did not significantly influence the expression of osteocyte markers. On the other hand, addition of Matrigel to the culture matrix significantly enhanced the expression of Rankl and late osteocyte markers such as Sost and Fgf23. Matrigel addition also inhibited upregulation of Opg and early osteocyte markers such as Dmp1 and Gp38. These effects on osteocyte properties were maximal at a Matrigel culture matrix content of 50 %. Matrigel addition to the matrix also increased dendritic process extension by osteocytes. In addition, Matrigel addition significantly stimulated tartrate-resistant acid phosphatase activity in co-culture with bone marrow macrophages. Among the conditions tested, 50 % Matrigel and 0.2 % FBS in type I collagen matrix were optimal for culture of primary osteocytes.

Organic cation transporter/solute carrier family 22a is involved in drug transfer into milk in mice.

Drug transfer into milk is a general concern during lactation. So far, breast cancer resistance protein (Bcrp) is the only transporter known to be involved in this process, whereas participation of other transporters remains unclear. We investigated the importance of organic cation transporter (Oct) in drug transfer into milk in mice. The mammary glands of lactating versus nonlactating FVB strain mice revealed elevated mRNA levels of Oct1 and Bcrp, whereas Oct2 and Oct3 mRNA levels were decreased. Specific uptake of cimetidine, acyclovir, metformin, and terbutaline was observed in human embryonic kidney 293 cells transfected with murine Oct1 or Oct2. The milk-to-plasma concentration ratio (M/P) values of cimetidine and acyclovir were significantly decreased in Bcrp knockout and Oct1/2 double-knockout (DKO) mice compared with control FVB mice, whereas the M/P values of terbutaline and metformin were significantly decreased in Oct1/2 DKO mice alone. These are the first to suggest that Oct1 might be involved in secretory transfer of substrate drugs into milk.

Hydroxyproline, a serum biomarker candidate for gastric ulcer in rats: a comparison study of metabolic analysis of gastric ulcer models induced by ethanol, stress, and aspirin.

Gastrointestinal symptoms are a common manifestation of adverse drug effects. Non-steroid anti-inflammatory drugs (NSAIDs) are widely prescribed drugs that induce the serious side effect of gastric mucosal ulceration. Biomarkers for these side effects have not been identified and ulcers are now only detectable by endoscopy. We previously identified five metabolites as biomarker candidates for NSAID-induced gastric ulcer using capillary electrophoresis-mass spectrometry (CE-MS)-based metabolomic analysis of serum and stomach from rats. Here, to clarify mechanism of changes and limitations of indications of biomarker candidates, we performed CE-MS-based metabolomic profiling in stomach and serum from rats with gastric ulcers induced by ethanol, stress, and aspirin. The results suggest that a decrease in hydroxyproline reflects the induction of gastric injury and may be useful in identifying gastric ulcer induced by multiple causes. While extrapolation to humans requires further study, hydroxyproline can be a new serum biomarker of gastric injury regardless of cause.

Regulatory mechanisms of RANKL presentation to osteoclast precursors.

It is important to understand the molecular mechanisms regulating osteoclast formation, as excess activation of osteoclasts is associated with various osteopenic disorders. Receptor activator of nuclear factor kappa B (RANKL) is a central player in osteoclastogenesis. Recent findings suggest that osteocytes are the major supplier of RANKL to osteoclast precursors. It has also been suggested that osteocyte cell death upregulates the RANKL/osteoprotegerin (OPG) ratio in viable osteocytes adjacent to apoptotic osteocytes in areas of bone microdamage, thus, contributing to localized osteoclast formation. Indeed, viable osteocytes can provide RANKL through direct interactions with osteoclast precursors at osteocyte dendritic processes. In addition, OPG tightly regulates RANKL cell surface presentation in osteocytes, which contributes to the inhibition of RANKL signaling, as well as the decoy receptor function of OPG. By contrast, the physiological role of RANKL in osteoblasts is yet to be clarified, although similar mechanisms of regulation are observed in both osteocytes and osteoblasts.