The appearance of immune checkpoint inhibitors has been a major turning point in cancer therapy. The success of immune checkpoint therapy has revolutionized the field of cancer therapy, and immunotherapy has joined the cancer treatment ranks as a pillar. To induce effective anti-tumor immune responses, it is necessary both to enhance the activity of immune cells and to block immune suppression by tumor cells. Carrier type drug delivery systems based on nanobiotechnology (nano DDS) represent a potentially useful technology for efficiently achieving both: enhancement of the activity of immune cells and blocking immune suppression. It has become clear that nano DDS can improve the practical utility of a wide variety of immune functional molecules and thus regulate drug kinetics and intracellular dynamics to improve drug efficacy and reduce side effects. We have been in the process of developing a nano DDS for the enhancement of cancer immunotherapy. A nano DDS encapsulating an agonist of a simulated interferon gene pathway greatly enhanced the activity of the agent's antitumor immune response. To block immune suppression, we successfully developed a small interfering RNA loaded into a nano DDS which regulates gene expression in immune cells. In this review, we summarize our recent efforts regarding cancer immunotherapy using nano DDS.
Silurus asotus egg lectin (SAL) is an α-galactoside-binding protein, isolated from the egg of catfish. It belongs to the rhamnose-binding lectin family that binds to Gb3 glycan (Galα1-4Galβ1-4Glc). SAL has resulted in the induction of early apoptosis in the Raji cell line, which is a Burkitt's lymphoma cell line expressing Gb3. The apoptosis was characterized by i) increased externalization of phosphatidylserin via multidrug resistance 1 P-glycoprotein (MDR1 P-gp), and ii) reduced cell size through the activation of voltage-gated potassium channel Kv1.3. Although the incorporation of propidium iodide (PI) was observed, SAL did not cause apoptosis in Raji cells. This event may be due to an increased expression of membrane-anchored tumor necrosis factor α (TNFα) and TNF receptor 1 (TNFR1) after the binding of SAL to Gb3. Moreover, SAL arrested the cell cycle at the G0/1 phase, thus inhibiting cell proliferation. The suppression of cell proliferation by SAL was likely due to the enhanced expression of p21 caused by the phosphorylation of ERK1/2 through the Ras-MEK-ERK1/2 pathway. Combination of SAL with anti-cancer drugs was also examined in this study. Interestingly, SAL increased the incorporation of doxorubicin (Dox) into Raji cells, consequently enhancing its cytotoxic effect. Similarly, the cytotoxic effects of vinblastine and irinotecan were also significantly increased in Raji cells treated with SAL. These studies demonstrate that SAL may be applied to cancer therapy.
Resistance to the breakpoint cluster region-abelson (BCR-ABL) tyrosine kinase inhibitor (TKI), imatinib, poses a major problem in the treatment of chronic myeloid leukemia (CML). Imatinib resistance often results from a secondary mutation in BCR-ABL1. However, the basis of this BCR-ABL1-independent resistance in the absence of such mutation remains to be elucidated. The aim of the present study is to identify the mechanism of imatinib resistance in CML. To gain insight into BCR-ABL1-independent imatinib resistance mechanisms, we performed an array-based comparative genomic hybridization. We identified various resistance-related genes, focusing on the receptor tyrosine kinase MET. Treatment with an MET inhibitor resensitized K562/IR cells to BCR-ABL TKIs. A treatment combining imatinib and a MET inhibitor in K562/IR cells inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation, but did not affect AKT activation. Moreover, the combination of MET inhibitor and imatinib suppressed tumor growth in vivo. These results indicate that the activation of MET/ERK and MET/JNK are potential mechanisms of BCR-ABL TKI resistance. Our findings provide new and important information concerning the mechanisms of imatinib resistance in CML, and reveal new proteins potentially involved in BCR-ABL TKI resistance.
Nasal application of powder formulations has garnered attention because of its significant potential for systemic drug delivery. Because a powder drug must first diffuse from the formulation and dissolve in the nasal cavity fluid before transepithelial permeation, dissolution and diffusion are distinct but important factors for nasal drug absorption. Since the formulation is directly administered onto the nasal mucosal surface, the effect of excipients on drug absorption may be significant. Therefore, the influence of excipients on nasal drug absorption was evaluated. Three types of hydroxypropyl cellulose (HPC) [HPC (SL), HPC (M), and HPC (H)], lactose, and sodium chloride (NaCl) were used as excipients. Warfarin (WF), piroxicam (PXC), sumatriptan (STP), and norfloxacin (NFX) were selected as model drugs. HPC (M) enhanced the absorption of PXC, while both HPC (M) and HPC (H) enhanced the absorption of STP. All three HPCs failed to enhance the absorption of WF. An increase in the polymerization degree of HPCs decreased the diffusion of drugs in HPC solutions, but prolonged their nasal retention. Lactose and NaCl increased the fluid volume on the nasal mucosal surface by increasing the osmotic pressure, thereby enhancing the nasal absorption of PXC and NFX; however, lactose and NaCl accelerated the nasal clearance of these. These results indicate that nasal drug absorption from powder formulations can be controlled by excipients.
In the development of quality biopharmaceutical drugs, it is the level of knowledge gained, not the volume of data, which provides the basis for science-based submissions and their regulatory evaluation [International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q8 (R2)]. The identification of critical quality attributes (CQAs) is important as the initial step for quality by design (QbD) strategies. The impact of each potential critical attribute (pCQA) is assessed by using a systematic and scientific approach with respect to bioactivity, pharmacokinetics (PK)/pharmacodynamics (PD), immunogenicity, and safety of the biopharmaceutical drugs. The purpose of this section is to introduce a strategy for identifying CQA, and new analytical methods for CQA assessment.
Therapeutic Fc-fusion proteins, created by linking bioactive peptides or receptor proteins to the Fc moiety of IgG, are currently being developed. In this development process, a Gly-Gly-Gly-Ser linker (G4S linker) is often used to link the peptide/protein and the Fc portion. O-xylose-type core glycans of glycosaminoglycan are known to attach to the Ser residue on the GSG motif in the G4S linker peptide repeats of the Fc fusion protein produced using the Chinese hamster ovary (CHO) cell expression system. In addition, a recent report demonstrated that unexpected mucin-type O-glycosylations occurred on a peptide in a bioactive peptide-Fc fusion protein; this glycosylation affected the bioactivity of the peptide. Therapeutic proteins with non-natural structures, such as Fc-fusion proteins, undergo unintended O-glycosylations; therefore, it is increasingly important to conduct detailed O-glycosylation analysis of fusion proteins during the developmental stages. In this paper, we have summarized recent reports on the unexpected O-glycosylation in fusion proteins, general O-glycosylation types and sequence motifs, and O-glycosylation analytical techniques involving O-linked oligosaccharide analysis and site-specific O-glycosylation analysis using LC/MS. In addition, we have introduced site-specific O-glycosylation analysis of Fc-fusion proteins with GS linker peptides by LC/MS using higher-energy collisional dissociation-tandem mass spectrometry (HCD-MS/MS) and electron-transfer dissociation (ETD)-MS/MS to obtain preferential dissociation of the peptide moiety in the glycopeptide.
Detailed structural characterization of protein biopharmaceuticals is a critical step in research and development; however, this step is often hampered by the structural complexities associated with glycosylation. Most protein biopharmaceuticals are modified with structurally heterogeneous and dynamic oligosaccharides which govern the physicochemical properties, functionality, pharmacokinetics, and potential pathogenicity of these glycoproteins. Considering this, we have developed a structural biological approach to describe the dynamic three-dimensional structures and interactions of glycoproteins as biopharmaceuticals. We developed an NMR technique assisted by metabolic stable-isotope labeling that can provide useful atomic-level probes for detecting and characterizing structural perturbations of glycoproteins caused by alterations in solution conditions and production protocols, as well as by mutagenesis. We have applied this method in conjunction with X-ray crystallography to investigate the structural impacts of varying glycoforms of the Fc region of immunoglobulin G (IgG), thereby elucidating the functional roles of the Fc glycans. In particular, we have successfully elucidated the structural mechanisms by which defucosylation of the IgG-Fc region increases its affinity for Fcγ receptor IIIa, leading to an improvement in ameliorating antibody-dependent cell-mediated cytotoxicity. In addition, we applied our stable-isotope-assisted NMR method to analyzing biomolecular interactions in serum environments, which are characterized by molecular crowding and promiscuous intermolecular interactions. An integrative structural biological approach combining NMR spectroscopy, X-ray crystallography, neutron scattering, atomic force microscopy, and molecular dynamics simulation will provide new research tools that will enable the visualization of dynamic structures and interactions of glycoproteins of pharmaceutical interest, thereby providing valuable insights for the development of biopharmaceuticals.
Biopharmaceuticals are often formulated as liquid dosage forms. During manufacturing and storage, protein molecules and active pharmaceutical ingredients form aggregates due to various stresses, including shaking and agitation, as well as by contact with silicone oils coated on pre-fillable syringes. The diameter of protein aggregates ranges from 15-20 nm, and that of dimers comprising a large number of antibody molecules can be up to 100 μm. Among these aggregates, those with a diameter of <100 nm are called nanometer aggregates, while those ranging between 100 nm and 1 μm are called sub-micron aggregates, and those ranging between 1 and 100 μm are called micron aggregates. In the last ten years, aggregates have been studied to determine their physical characteristics and their impact on immunogenicity. As a result, novel analytical methods and instruments for such characterizations have been established for a majority of aggregates, including those that are difficult to evaluate. Here, the biophysical features of protein aggregates are explained, followed by an introduction to the different methods for aggregate characterization, including their advantages and actual results. Finally, future perspectives and expectations regarding the characterization of protein aggregates are proposed.
Although many people (and patients) in Japan currently consume health foods such as supplements, few have proper knowledge of their usefulness and safety. In December 2015, the Food Safety Commission of Japan issued a report and 19 messages mainly on the safety of health foods to disseminate appropriate knowledge to consumers. The report divided health food risks into three categories: 1) risks as food (e.g., increased lung cancer risk in smokers consuming excess β-carotenoid); 2) risks as health foods (e.g., short consumption history, drug contamination, poor quality of active ingredients, and interactions with drugs); and 3) risks due to a lack of adequate scientific information on health foods. The risk of insulin autoimmune syndrome caused by α-lipoic acid is relatively high among Japanese individuals because its onset is associated with HLA-DRB1*04:06, an HLA allele occurring frequently in East Asian populations. As for health food-drug interactions, an important pharmacokinetic interaction between drugs and St. John's Wort was described from several viewpoints: different effects on drugs within the same class (depending on the metabolic pathway); interindividual differences in its effects; importance of considering active metabolite involvement; and time course of interaction. An example of an interaction affecting drug efficacy was also introduced. Because the Japanese government now promotes a health-supportive pharmacy program in which pharmacies have a role in supporting the health of local patients/consumers, pharmacists are expected to acquire more scientific information on health foods, evaluate their evidence levels, and provide that information in plain language to patients/consumers.
Along with an increase in self-care and self-medication practices, the use of health foods as primary and secondary methods of disease prevention has increased. Consumers are aware only of the health benefits of dietary ingredients, although the potential risks associated with most ingredients are unknown. Adverse events associated with the use of health foods have been reported, and in some cases they were due to inappropriate use such as the concomitant use of several health foods or health foods and drugs. It is important that healthcare professionals, especially pharmacists, provide reliable, evidence-based information to ensure the safe and appropriate use of dietary supplements by their patients. Thus, we constructed an online database, the “Health Foods Network (HFNet)” that compiles reports on the safety and effectiveness of health foods and their ingredients. It serves to disseminate information based on scientific research not only in Japan but also worldwide. This article provides an overview of the HFNet. Additionally, findings from our recent survey and educational interventions among college students are discussed. We hope that this article will be helpful for pharmacists and other healthcare professionals who provide consultations on the use of health foods.
We have been studying the way advertisements for medicines have been monitored by the Health, Labour and Welfare Sciences Research Grants. In the last fiscal year, we identified products that were being advertised to general consumers, such as OTC drugs and designated quasi-drugs, and made recommendations to the Ministry of Health, Labour and Welfare on the methods of advertisement. Members of our research group, including physicians, pharmacists, and consumers, pointed out that advertisements for so-called health foods and foods with functional claims were much more unregulated than those for drugs, including OTC drugs. Thus, this fiscal year, we decided to focus on health foods. Advertisements for health foods are regulated by the Health Promotion Act and the Act against Unjustifiable Premiums and Misleading Representations; moreover, in many cases, cautions are issued by the Consumer Affairs Agency or prefectures. Several studies also reported health damage in patients who, because of their belief in advertisements, intentionally discontinued treatment or missed the opportunity to receive appropriate medical treatment. Here, we report inappropriate advertisements for health foods that may cause health hazards.
In recent years, consumer interest in health and health foods (e.g., dietary supplements) has increased, and the types of and market for health foods have also expanded rapidly. The safety of health foods is a significant concern in many countries. Numerous adverse events associated with the consumption of health foods have been reported. There are cases that have resulted in serious liver failure, renal failure, and death in other countries. These products may contain undeclared medical ingredients and toxic chemical compounds that are illegally and intentionally added or contain natural plant toxins. Products containing high levels of heavy metals have also been reported to cause such adverse events. Some products remain on the market, even after regulatory agencies alert consumers and issue warnings to their sellers. Moreover, because people can buy health foods from sources overseas via the Internet, adverse effects associated with the use of such products remain a concern. Two cases of adverse events were reported in Japanese individuals who purchased “OxyElite Pro” products imported privately. They are advertised as weight-loss and muscle-building products and have been associated with many cases of liver failure and hepatitis in the USA. In this paper, regulatory systems and adverse events associated with the use of health foods in other countries are discussed.
We previously showed that a naturally occurring macrocyclic bis(bibenzyl) derivative, riccardin C (RC), exhibits antibacterial activity towards methicillin-resistant Staphylococcus aureus (MRSA), with a potency comparable to that of the clinically used drug vancomycin. Here, we synthesized a series of RC derivatives to explore the structure-activity relationships (SAR). The SAR results clearly indicated that the number and positions of the phenolic hydroxyl groups are primary determinants of the anti-MRSA activity. Pharmacological characterization of the macrocyclic bis(bibenzyl) derivatives, together with fragment compounds and their dimers, indicated that the macrocycles and the fragment compounds elicit anti-MRSA activity with different mechanism(s) of action. The macrocyclic bis(bibenzyl)s are bactericidal, while the fragment compounds are bacteriostatic, showing only weak bactericidal activity. Treatment with a macrocyclic bis(bibenzyl) derivative significantly changed the intracellular Na+ and K+ concentrations of Staphylococcus aureus, and transmission electron microscopy revealed that treated cells developed intracellular lamellar mesosomal-like structures. These results indicated that the macrocyclic compound directly damages the gram-positive bacterial membrane, resulting in increased permeability.
We showed that a naturally occurring macrocyclic bis(bibenzyl) derivative, riccardin C (RC) and some synthetic derivatives, exhibits antibacterial activity towards MRSA, with a potency comparable to that of the clinically used drug vancomycin. The SAR results indicated that the number and positions of the phenolic hydroxyl groups are primary determinants of the anti-MRSA activity. Numerous biological studies indicated that the macrocyclic bis(bibenzyl) derivatives directly damages the gram-positive bacterial membrane, resulting in increased permeability.
Maintaining medication adherence is a critical issue in determining health outcomes in patients with chronic diseases. However, many patients do not adhere to their prescribed regimens. This study aimed to determine the effects of using adherence score sheets according to application timing in improving medication adherence among non-adherent outpatients. In community pharmacies, both patients and pharmacists evaluated medication adherence based on application timing (morning, noon, evening, and before going to bed) in 11 levels (0-10) for >4 months. A total of 58 outpatients were included in the study. The median scores among application timing at intermediate (patient 9.3, pharmacist 9.0) and final (patient 9.5, pharmacist 9.5) analyses were significantly higher than that at baseline (patient 7.6, pharmacist 7.0). At the end of the investigation, the ratio of non-adherent patients prescribed with hyperlipidemic medications was higher than those prescribed with medications for other lifestyle diseases. Approximately 80% of the patients reported improved medication adherence based on the questionnaires regarding their understanding on diseases and medications, medication awareness, and communication with pharmacists. Therefore, the utilization of an adherence score sheet according to application timing improved medication adherence of patients with chronic diseases.
Hospital formulation has several advantages, including the flexibility of customization as per the disease state or the patients' precise requirements. However, compared with commercial formulations, hospital formulations are usually not under the same level of quality check. In the present study, we tested mixed powder formulations prepared in a hospital pharmacy using Raman spectroscopy to investigate the feasibility of applying Raman spectroscopy as a quality-control tool of hospital formulations. For this purpose, we first established a numerical evaluation method to determine the uniformity of a powder mixture using Raman chemical imaging data with atropine sulfate/lactose mixture samples and revealed that the mixing uniformity correlated to the experience level of the pharmacist. Next, we developed a content quantification method in a one-dose packaged powder formulation by measuring the Raman spectra from the outside of the package. Because this method allows for quantification of the components inside the package in a non-destructive and non-contact manner, it can be applied for content confirmation after one-dose packaging. Using this method, the content uniformity of the mixed powder formulation in the one-dose package was compared between the formulations prepared by the pharmacists and those prepared by a pharmacy robot. Our study indicates the possibility of applying Raman spectroscopy as a quality-control tool of hospital formulations. Studies on further applications of Raman spectroscopy in the field of clinical pharmacology are expected.
Volatile components originating from Lavandula angustifolia Hidcote and Lavandula x intermedia Grosso plants cultivated in a medicinal plant garden of Hoshi University located in southern Tokyo were investigated using thermal desorption-GC-MS. Sampling of the flowers and herbs of the lavender was performed at different developmental stages, i.e., summer, autumn, and winter (herbs only) using solid phase micro extraction fibers. Linalool, linalyl acetate, β-caryophyllene, β-myrcene, α-ocimene, β-ocimene, and terpinen-4-ol were the predominant constituents originating from the flowers of both plants. Additional volatile compounds such as borneol, eucalyptol, and camphor were found in the Lavandula x intermedia flowers. The number of volatile compounds originating from the Lavandula angustifolia flowers in summer was greater than that in autumn. 3-Hexen-1-ol, 3-carene, and p-cymen-8-ol were volatile compounds characteristic of the herbs, and α-ocimene, allo-ocimene, and terpinen-4-ol were detected only from flowers. In comparing volatile compounds obtained from fresh herbs with those from dried herbs, camphor, β-caryophyllene, and borneol were absent in dried herbs. For people who visit the plant garden, smelling the fragrances of plants directly is believed to deliver not only pleasure but also beneficial therapeutic properties.
Communication education is now necessary for pharmaceutical education since the role of pharmacists has expanded from “medicine-based” to “person-based”. However, a standard for assessing the effectiveness of a communication education program has not been established. Hence, the aim of this study was to determine the effectiveness of clinical training in pharmacy for enhancing the ability of pharmacy students to communicate. Role playing with simulated patients was performed by pharmacy students before and after clinical practice for pharmacy, and the effects of learning were analyzed by Roter method of interaction process analysis (RIAS). Analysis by RIAS enabled quantification and objective evaluation of communication by pharmacy students. The results showed improvement of interactive communication, decrease of “Question asking” and “Others” including “Transition words”, and increase of “Partnership behaviors” and “Counsel behaviors”. The pharmacy students became skillful in communication without showing hesitation. The results therefore showed that clinical training contributes to improvement in the ability of pharmacy students to communicate.
The aim of this study has been to investigate the time-to-onset and onset-pattern of drug-induced interstitial lung disease (DILD) after the administration of monoclonal antibodies through the use of the spontaneous adverse reaction reporting system of the Japanese Adverse Drug Event Report database. DILD datasets for adalimumab, bevacizumab, cetuximab, denosumab, golimumab, infliximab, nivolumab, panitumumab, pembrolizumab, tocilizumab, and trastuzumab were used to calculate the median time-to-onset of DILD, as well as the Weibull distribution parameters. The median time-to-onset of DILD for pembrolizumab and infliximab was within 1 month. The median time-to-onset of DILD for cetuximab, nivolumab, panitumumab, bevacizumab, golimumab, trastuzumab, and tocilizumab ranged from 1 to 2 months. The median time-to-onset of DILD for denosumab and adalimumab was more than 2 months. Infliximab, trastuzumab and tocilizumab, and denosumab were estimated to fit the early failure type profile of the Weibull distribution parameters. Cetuximab, nivolumab, panitumumab, bevacizumab, golimumab, and adalimumab were estimated to fit the random failure type profile. Pembrolizumab was estimated to fit the wear out failure type profile. Cluster analysis was performed to classify the time-to-onset patterns of DILD. Hierarchical cluster analysis showed 3 clusters. The findings of this study established both the most likely time period and onset-pattern of DILD that can occur in patients after the administration of monoclonal antibody agents.