Recently, the application of β-cyclodextrins (β-CDs) as therapeutic agents has received considerable attention. β-CDs have been reported to have therapeutic effects on various diseases, such as Niemann-Pick type C (NPC) disease, a family of lysosomal storage disorders characterized by the lysosomal accumulation of cholesterol. To further improve the therapeutic efficacy of β-CDs, the use of β-CD-threaded polyrotaxanes (PRXs) has been proposed as a carrier of β-CDs for NPC disease. PRXs are supramolecular polymers composed of many CDs threaded onto a linear polymer chain and capped with bulky stopper molecules. In this review, the design of PRXs and their therapeutic applications are described. To achieve the intracellular release of threaded β-CDs from PRXs, stimuli-cleavable linkers are introduced in an axle polymer of PRXs. The stimuli-labile PRXs can dissociate into their constituent molecules by a cleavage reaction under specific stimuli, such as pH reduction in lysosomes. The release of the threaded β-CDs from acid-labile PRXs in acidic lysosomes leads to the formation of an inclusion complex with the cholesterol that has accumulated in NPC disease patient-derived fibroblasts, thus promoting the extracellular excretion of the excess cholesterol. Moreover, the administration of PRXs to a mouse model of NPC disease caused significant suppression of the tissue accumulation of cholesterol, resulting in a prolonged life span in the model mice. Additionally, the induction of autophagy by the methylated β-CD-threaded PRXs (Me-PRXs) is described. Accordingly, the stimuli-labile PRXs are expected to be effective carriers of CDs for therapeutic applications.
We focused on the crystal structure of cyclodextrin (CD) to develop new solid CD complexes. There are two large spaces in the columnar structure of CD crystals: one inside a CD cavity and another between CD columns. New solid CD complexes can be designed by incorporating guest drugs between the CD columns. We succeeded in preparing a solid drug/[polyethylene glycol (PEG)/γ-CD-polypseudoraxane (PPRX)] complex by a sealed-heating method via the gas phase. A drug/(PEG/γ-CD-PPRX) complex has a structure in which PEGs are included in a γ-CD cavity, and guest drugs are incorporated between the γ-CD columns. Screening by a sealed-heating method determined that a variety of guest drugs with varying molecular size and log P could be incorporated into the spaces between γ-CD columns, following a stoichiometric rule. Another method, via solid phase using cogrinding and subsequent heating, was developed to prepare drug/(PEG/γ-CD-PPRX) complex. This method enabled us to prepare the complex with a thermally unstable drug, as well as a drug/(PEG/α-CD-PPRX) complex not formed using the sealed-heating method. Both the structure and molecular state of each drug in the complexes were characterized by powder X-ray diffraction and solid-state NMR measurements. The dissolution character and thermal stability of the drug incorporated in the complex could be improved by the specific complex formation. The solid CD complexes thus developed have potential for drug-encapsulation and as drug-release carriers, owing to their unique structural and pharmaceutical properties.
Molecular recognition is useful in creating functional supramolecular materials. Non-covalent bond formations, such as host-guest interactions, hydrogen bonding, and electrostatic interaction, are effective tools for introducing various functions and properties into materials. This review focuses on such macroscopic functions as selective molecular adhesion, self-healing, toughness, and the actuation of supramolecular polymeric materials—materials which have potential in pharmaceutical development. These functions have been achieved using reversible bonding between cyclodextrins (CDs; cyclic host molecules) and guest molecules. For example, macroscopic adhesions between host-modified hydrogels and guest-modified hydrogels have been investigated. CD-modified hydrogels were found to show selective adhesion to a guest hydrogel with an appropriate molecular size for the CD cavity, indicating that the host-guest complex formation between the gels led to the adhesive behavior. Surprisingly, polymeric materials having host-guest cross-linking points show both high toughness and flexibility, unlike conventional covalently cross-linked materials. These materials also exhibited self-healing properties, capable of repairing damage to the materials. Furthermore, the supramolecular materials demonstrated macroscopic rapid expansion and contraction driven by external stimuli under wet or semi-dry conditions, in which the supramolecular gels vary the cross-linking density between the polymers accordingly. Different topological gels are able to vary the length of the polymer chain between cross-linking points to show large deformation. Both types of actuators were found to exhibit externally stimulated flexing behaviors. This review summarizes recent advancements in the development of these supramolecular materials, which appear to be promising new components in pharmaceutical science.
Supramolecular chemistry is a useful and important domain for understanding pharmaceutical sciences, since various physiological reactions (e.g., protein association) and drug activities (e.g., the substrate/receptor reaction) are based on supramolecular chemistry. Biological components, such as DNA and cells, are also supermolecules. However, supramolecular chemistry to date has not been a major domain in the field of pharmaceutical study. In this article, we propose a new concept in pharmaceutical sciences termed “supramolecular pharmaceutical sciences” which combines pharmaceutical sciences and supramolecular chemistry. “Supramolecular pharmaceutical sciences” could encompass strictly controlled molecular arrangement, stimulus responsible molecular motion, new functions beyond those of existing molecules, more accurate drug design, new active pharmaceutical ingredients, new perspectives for the investigation of the drug mechanisms, and novel pharmaceutical technologies. Moreover, pharmaceutical sciences are useful for supramolecular chemistry, because biological reactions are very accurate reactions, making this a win-win relationship. Thus, supramolecular pharmaceutical sciences could be useful for developing new methods, hypotheses, ideas, materials, mechanisms, and strategies in the realm of pharmaceutical science.
Conducting research abroad can be an invaluable experience. I would like to share my own experiences as an example of how one's PhD course research (in the laboratory of Shibasaki Masakatsu) and research abroad (University of Wisconsin-Madison, laboratory of Laura Kiessling) can contribute to one's subsequent research. During my PhD course research in Japan, I investigated asymmetric catalysis in the efficient production of physiologically active substances. Then I moved to the U.S. and worked on the functional analysis of enzymes (biocatalysts), with a specific focus on glycosyltranspolymerase. Now I am working on the development of chemical catalysts that can introduce post-translational modifications into proteins, especially histones. Being based in catalysis, I have aimed at research that can contribute to the life sciences. In this account, I share my thought process on selecting such a career path, discuss how it affected my current research, and touch on the positive aspects as well as points for reflection when conducting research abroad.
Proteins are excellent materials for constructing nano- to micro-meter sized compartments. For example, in nature, hollow spherical shells made of proteins, called protein cages, are widespread. Prominent examples include viruses, ferritins, carboxysomes, and others. Protein cages designed and engineered in the laboratory have also gained recent attention because of their potential use in synthetic biology, materials science, and medicine. Here, we show that engineered variants of lumazine synthase (LS) from Aquifex aeolicus self-assemble into porous shell-like structures, with striking size-expansion from the original dodecahedron composed of 12 pentamer subunits. Cryo-electron microscopy (EM) analysis has revealed that pentamers are the basic assembly units, although small conformational changes in each subunit lead to final expanded architectures composed of 36 and 72 pentamers. The underlying conformational changes likely arise from electrostatic repulsion between anionic residues originally introduced at the lumenal surface of the LS cage to encapsulate positively charged guest molecules. The plastic nature of the LS cage structure was also explored using a positively supercharged variant of the green fluorescent protein GFP(+36) as an assembly mediator. By controlling the favorable electrostatic interactions between the negatively charged LS cage and the positively charged mediator, multishell structures were created, as previously observed in some virus-like particles. These results highlight the potential of engineered LS cages for various future applications, including drug delivery and bioimaging.
Antibody-drug conjugates (ADCs), monoclonal antibodies conjugated with highly potent drugs (payloads) through chemical linkers, are an emerging class of therapeutic agents for cancer chemotherapy. Their clinical success has been demonstrated by the 4 ADCs already approved by the U.S. Food and Drug Administration (FDA), and more than 60 promising ADCs now in clinical trials. Further advancement of this novel molecular platform could potentially revolutionize current strategies and regimens for treating cancers. The linker structure and antibody-linker conjugation modality critically contribute to ADC homogeneity, circulation stability, pharmacokinetic profiles, tolerability, and overall treatment efficacy. Despite extensive efforts to improve these parameters, most ADC linkers used to date possess linear structures, and therefore accommodate only single payloads. The clinical potential of branched ADC linkers, enabling the installation of two payload molecules, remains unexplored because of the lack of efficient conjugation methods. In addition, according to a recent report, the stability of enzymatically cleavable linkers in mouse circulation is another crucial factor for the successful evaluation of ADCs in preclinical studies. In this review, I present my research group's effort to develop both branched linkers and efficient conjugation methods for constructing dual-loading ADCs with high homogeneity and enhanced potency. I also present a novel tripeptide ADC linker with enhanced stability in mouse circulation. Multidisciplinary experience, approaches, and collaboration are key to successfully advancing our ADC research programs. I herein describe how my experience in the U.S. has helped to develop and manage complex biomedical research projects in a small academic laboratory setting.
Just after receiving my Ph.D. degree in 2004 from Tokushima University, under the supervision of Professor Masayuki Shibuya, I had the opportunity to work as a Research Associate in the laboratory of Professor Kim D. Janda at The Scripps Research Institute in the U.S., for about a year. Since it has already been more than 10 years since my time at Scripps, the specific research performed at that time may no longer be of interest to readers, but the benefit of working in a different research environment is timeless. Therefore, this paper describes not only details of the research conducted, but also the significance of working in a foreign country as a postdoc, and the subsequent influence those experiences at The Scripps Research Institute have had on my career.
Ten years ago, in April 2007, I went abroad to study at The Scripps Research Institute (TSRI) in San Diego, USA. As an overseas researcher with the Japan Society for the Promotion of Science (JSPS), I worked with Professor Phil S. Baran (an associate professor at the time), who was a distinguished young researcher in synthetic organic chemistry. Working abroad had been my dream ever since I had decided to work at a university as a researcher. Through my study of organic chemistry, I hoped to spread my wings and explore the world. Fortunately, the research projects at TSRI went well, and a year and a half later, I returned to Japan, to Nagoya University as an assistant professor (under the guidance of Professor Kenichiro Itami). During my time abroad, I certainly gained a lot of experience in chemistry, but as I look back to 10 years ago, I feel that the personal interactions remain much more important to me. Numerous Japanese researchers who studied overseas around the same period, and many TSRI graduate students I encountered, are currently faculty members at top universities around the world. At this memorable and nostalgic phase of my life, I am sharing here a personal account of the research I conducted and the researchers I met during my stay in San Diego.
Dynamic light scattering (DLS) is used for measuring average particle diameter and particle diameter distribution of nano-sized particles dispersed in liquid. The parameters are important characteristics mainly for drug delivery system (DDS) formulations, such as solid in oil (S/O) formulations, liposome formulations, suspension formulations, and emulsion formulations. Herein are described a measurement method and measurement case of particle diameter analysis of medical products using laser light.
Over the past few decades, liposome drug delivery systems (liposome DDS) have attracted much attention as the most advanced DDS. Efficacy and toxicity profiles of liposomes are based on their characteristic pharmacokinetics, drug release, and disposition after administration. Many attempts have been made to develop these systems especially as liposomal anti-cancer drugs. In the development of liposome DDS, identification of critical quality attributes and establishment of a control strategy to ensure consistent drug product quality are crucial. Among the quality attributes, particle size, drug encapsulation, and drug release from liposomes would affect their in vivo pharmacokinetic and pharmacodynamic properties. Thus these features need to be evaluated with appropriate analytical methods to confirm the quality and performance of the drug products. This article focuses on drug release from liposomes and reviews the effects of physicochemical properties of loaded drugs on release, simulation of drug release from liposomes, and design of a simulated body fluid for drug release assay of drug products.
Nanotechnology-based formulations have attracted much attention as delivery tools for a variety of payloads. These payloads include oligonucleotides, peptides, and low-molecular weight chemical entities. Guidelines and reflection papers for nanotechnology-based drug products have been published by the Japanese Ministry of Health, Labour, and Welfare. These documents include the concept of quality by design (QbD) approach, as described in the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The quality attributes that affect efficacy and safety of nanotechnology-based drug products should be identified to establish the entire picture of the drug products. Hence it is essential to develop analytical methods for characterizing these attributes. Furthermore, to evaluate the comparability of nano-drug delivery system (DDS) formulations before and after changes to their manufacturing process, it is desirable to characterize the key attributes using more than one analytical method for each. Standardization of these analytical methods is underway. This paper provides an overview of the concept and significance of the QbD approach for nano-DDS formulations, guidelines for the development of nano-DDS formulations, and standardization of analytical methods for nano-DDS formulations.
The development of novel photolabile protecting groups with practical levels of photolytic efficiency and hydrophilicity can provide smart photochemical tools, such as caged compounds. One of the long-standing problems of most reported photolabile protecting groups is the requirement for one-photon activation, of ultraviolet light (250-400 nm), that is harmful to living cells and has low tissue penetration power. An attractive approach to overcome this would be the use of longer-wavelength light for one-photon activation; advantages would include both lower phototoxicity and higher tissue penetration power than UV irradiation. As part of our research aimed at developing new photochemical tools, we have developed the N-methyl-7-hydroxyquinolinium (N-Me-7-HQm) caging chromophore as a novel photocage, sensitive to visible light. A key to the success of the development of the N-Me-7-HQm photocage was simple N-methylation of the 7-hydroxyquinoline chromophore. This modification allows access to visible light absorbance, facile photoactivation by blue-LED light (458 nm) with high photolytic efficiency, excellent water solubility, and high resistance to spontaneous hydrolysis. The success of the late stage upgrading of a chromophore in the synthetic sequence suggests that further functionalization of the caging chromophore will be possible, and should aid in the rapid generation of structurally diverse libraries of visible light-sensitive photocages.
Detecting the behaviors of proteins in membranes is often challenging; we need to develop new methods to better understand the mechanisms involved. We have developed two types of peptide-based experimental systems that can detect the self-association of proteins in bilayer environments: 1) a single-pair fluorescence detection system for studying the self-association of transmembrane helices in model membranes; and 2) live-cell fluorescence labeling and analysis of the oligomeric state of membrane proteins using a coiled-coil labeling method. By using these methods, we show that membrane cholesterol significantly affects the self-association of transmembrane helices.
Rhodamine dyes are among the most widely used fluorescent dyes for bioimaging due to their high fluorescence quantum yield and high photostability. Recently, novel far-red to near-infrared (NIR) fluorescent dyes have been developed: Si-rhodamines (SiRs), in which the O atom of conventional rhodamine dyes at the 10 position of the xanthene moiety is replaced with a Si atom. These SiRs are excellent long-wavelength fluorophores for bioimaging, as they retain the advantageous photophysical properties of conventional rhodamine dyes. Further, we focused on the QSY dark quenchers, which contain the rhodamine scaffold bearing aromatic rings at the N atoms at the 3,6-positions of the xanthene moiety; these show no fluorescence, irrespective of solvent polarity and pH. NIR fluorescent probes based on the Förster resonance energy transfer (FRET) mechanism have various practical advantages, and their molecular design is generally based on the use of NIR dark quenchers as cleavable FRET acceptors. However, few NIR dark quenchers can quench fluorescence in the Cy7 region (over 780 nm). We successfully developed SiR-based NIR dark quenchers (SiNQs) which show broad absorption covering this region. To demonstrate their usefulness, we designed and synthesized a NIR fluorescence probe for matrix metalloproteinase (MMP) activity using SiNQs. The developed probe was able to detect MMP activity in terms of NIR fluorescence, not only in vitro, but also in cultured cells and in a tumor-bearing mouse, in which the tumor was clearly visualized.
This review reflects back over almost 40 years of the author's basic research conducted at Graduate School of Pharmaceutical Sciences, Osaka University, Japan. After performing postdoctoral research in USA, the author became a research associate at Prof. Yoshiharu Miura's lab and started research on Biochemical Engineering in 1984. At that time, the main research purpose was to solve global environmental issues for maintaining human health. The author's achievements included novel useful material production system under inorganic conditions and genetically engineered whole-cell bacterial sensors detecting arsenite by naked eye without a detecting device. Another theme in the lab was to construct bioartificial liver support system. Various scaffolds for hepatocytes were newly prepared for constructing the compact reactor. Besides the bioreactor study, the author conducted cell transplantation research for the treatment of chronic liver diseases. It was shown that mesenchymal stem cells derived from third molars (wisdom teeth) could differentiate into hepatocytes and exhibit therapeutic effects in liver-damaged animals. After 2006, the lab started research on drug delivery systems, including noninvasive delivery of drugs such as peptides and nucleic acids by regulating epithelial tight junctions. Many substances enabling drug delivery through “paracellular” route were newly prepared. The author started basic research on Biochemical Engineering in the 1970s. Although these studies eventually shifted into the pharmaceutical field, the underlying concept was based on “engineering” throughout a 40-year research period. The author cordially thanks all colleagues for supporting engineering research in our lab.
To clarify the volume of water required to paste pediatric powders, we herein established a standard for the powder paste state by measuring yield values when water was added to powders. The powders used in the present study were selected from 8 types including original and generic drugs. Tipepidine hibenzate is prescribed in the pediatric field in combination with ambroxol hydrochloride and l-carbocysteine. The volumes of water needed to achieve the paste state of ambroxol hydrochloride between the original and generic drugs were similar. However, the volumes of water needed for l-carbocysteine markedly differed between the original and generic drugs due to differences in their additives. The spreadability of the mixture when water was added to the powders was evaluated using a spread meter. Among the powders tested in the present study, the yield value to achieve a paste state with the addition of water was approximately 1000 dyne/cm2. The optimum volume of water estimated from this yield value using the linear proportional relationship for the amount of powder may be applied to the mixture of each pediatric power for dosage/body weight.
To clarify the volume of water required to paste pediatric powders, we herein established a standard for the powder paste state by measuring yield values when water was added to powders. The yield value to achieve a paste state was estimated approximately 1,000 dyne/cm2 from the spreadability using a spread meter. The optimum volume of water estimated from this yield value for the amount of powder may be applied to the mixture of each pediatric power for dosage/body weight.
We developed a flow injection analysis (FIA) method based on tris(2,2′-bipyridine)ruthenium(III) [Ru(bpy)3 3+] electrogenerated chemiluminescence for assessment of antioxidant property. The hydroxyl radical (∙OH) were generated by H2O photolysis using an ultraviolet/H2O photoreactor. The reactor comprised a polytetrafluoroethylene tube, a quartz container, and a low-pressure mercury lamp that predominantly emitted radiation at around 185 nm. When the hydroxyl radical and Ru(bpy)3 3+ were in contact, chemiluminescence was generated as background emission. The background emission decreased when antioxidant samples were injected to the system. The antioxidant property of the naturally occurring antioxidants tested are listed herein, starting with the highest: gallic acid>ascorbic acid>quercetin. Moreover, our method allowed a sample throughput of approximately 100 samples/h. The proposed high throughput method can be used to assess the antioxidant property of the naturally occurring antioxidants.
Psychiatric treatment is shifting from hospital to ambulatory care. It is important that pharmacists positively support outpatients. Pharmacist-led interviews with outpatients have been conducted in the psychiatric department of Iizuka Hospital before examination by the doctor since 2015. Few studies in this field have reported about the effect of the pharmacist-led interviews using subjective evaluation of outpatients prior to examination by doctors. The aim of this study was to reveal this effect by the evaluation of outpatients. We conducted a questionnaire survey. More than 80% of the patients responded that it was “Good” to have an interview with the pharmacist prior to examination by the doctor. Moreover, 71.7% of the patients were “Satisfied” with the pharmacist-led interview, while 81.7% of them responded to “Agree” about continuing the interview in the future. Patients who were satisfied and wished to continue the pharmacist-led interviews were more likely to report better rapport with the doctor as well, in comparison to the patients who answered negatively. Furthermore, the patients who answered “Satisfied” were significantly less likely to forget reporting to the doctor than those who answered negatively. The pharmacist-led interviews in the psychiatric department were appreciated by the patients. In conclusion, pharmacists can facilitate communication between patients and doctors through these interviews. These results indicate that the pharmacist-led interview before the doctor examination is a useful effort from the perspective of outpatients.
Construction of regional inclusive care system is being promoted and pharmacists are required to implement multidisciplinary collaboration in order to provide appropriate pharmaceutical care to patients. However, there are few studies on collaboration between medical facilities and community pharmacies. In this study, we conducted a questionnaire survey of community pharmacies in Gifu City, which assessed the experience and attitudes regarding collaboration with other medical facilities. The survey items are: I. Participation in regional care meetings, II. Case discussion conferences, III. Joint workshops/study conferences, IV. Community service, V. Sharing information through medical cooperation network, and VI. Accompanying community pharmacists at home medical care. For the implementation of collaboration, the percentage of “not implemented” were as high as 70% or more in II, IV, V and VI. Regarding the attitudes toward collaboration, more than half of pharmacies answered that they wanted to implement in all items. In the comparison by the number of pharmacists, pharmacies with two or more pharmacists had significantly higher implementation ratios than pharmacies with one pharmacist in IV and V. Regarding the attitudes toward collaboration, pharmacies with two or more pharmacists had significantly higher ratios of considering implementation than pharmacies with one pharmacist in I and VI. Based on the results of this survey, there were many items that were not implemented as collaboration with other medical facilities at community pharmacies. However, many community pharmacies are planning to collaborate with other medical facilities in the future.