Prodrug Activation by Gold Artificial Metalloenzyme-Catalyzed Synthesis of Phenanthridinium Derivatives via Hydroamination.

An emerging approach in the field of targeted drug delivery is the establishment of abiotic metal-triggered prodrug mechanisms that can control the release of bioactive drugs. Currently, the design of prodrugs that use abiotic metals as a trigger relies heavily on uncaging strategies. Here, we introduce a strategy based on the gold-catalyzed activation of a phenanthridinium-based prodrug via hydroamination under physiological conditions. To make the prodrug strategy biocompatible, a gold artificial metalloenzyme (ArM) based on human serum albumin, rather than the free gold metal complex, was used as a trigger for prodrug activation. The albumin-based gold ArM protected the catalytic activity of the bound gold metal even in the presence of up to 1 mM glutathione in vitro. The drug synthesized via the gold ArM exerted a therapeutic effect in cell-based assays, highlighting the potential usefulness of the gold ArM in anticancer applications.

Artificial Glycoproteins as a Scaffold for Targeted Drug Therapy.

Akin to a cellular "fingerprint," the glycocalyx is a glycan-enriched cellular coating that plays a crucial role in mediating cell-to-cell interactions. To gain a better understanding of the factors that govern in vivo recognition, artificial glycoproteins were initially created to probe changes made to the accumulation and biodistribution of specific glycan assemblies through biomimicry. As a result, the organ-specific accumulation for a variety of glycoproteins decorated with simple and/or complex glycans was identified. Additionally, binding trends with regard to cancer cell selectivity were also investigated. To exploit the knowledge gained from these studies, numerous groups thus became engaged in developing targeted drug methodologies based on the use of artificial glycoproteins. This has either been done through adopting the glycoprotein scaffold as a drug carrier, or to directly glycosylate therapeutic proteins/enzymes to localize their biological activity. The principle aim of this Review is to present the foundational research that has driven artificial glycoprotein-based targeting and subsequent adaptations with potential therapeutic applications.

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers.

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2-10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by 31P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2-3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.

The Perpendicular Orientation of Amphotericin B Methyl Ester in Hydrated Lipid Bilayers Supports the Barrel-Stave Model.

The clinically important antibiotic amphotericin B (AmB) is a membrane-active natural product that targets membrane sterol. The antimicrobial activity of AmB is generally attributed to its membrane permeabilization, which occurs when a pore is formed across a lipid bilayer. In this study, the molecular orientation of AmB was investigated using solid-state nuclear magnetic resonance (NMR) to better understand the mechanism of antifungal activity. The methyl ester of AmB (AME) labeled with NMR isotopes, d3-AME, and its fluorinated and/or 13C-labeled derivatives were prepared. All of the AmB derivatives showed similar membrane-disrupting activities and ultraviolet spectra in phospholipid liposomes, suggesting that their molecular assemblies in membranes closely mimic those of AmB. Solid-state 2H NMR measurements of d3-AME in a hydrated membrane showed that the mobility of AME molecules depends on concentration and temperature. At a 1:5:45 AME:Erg:dimyristoylphosphatidylcholine ratio, AME became sufficiently mobilized to observe the motional averaging of quadrupole coupling. On the basis of the rotational averaging effect of 19F chemical shift anisotropy, 2H quadrupolar splitting, and 13C-19F dipolar coupling of 14ß-F-AMEs, we deduced that the molecular axis of AME is predominantly parallel to the normal of a lipid bilayer. This result supports the barrel-stave model as a molecular assembly of AmB in membranes.

The Amphotericin B-Ergosterol Complex Spans a Lipid Bilayer as a Single-Length Assembly.

Amphotericin B (AmB) is a polyene macrolide antibiotic clinically used as an antifungal drug. Its preferential complexation with ergosterol (Erg), the major sterol of fungal membranes, leads to the formation of a barrel-stave-like ion channel across a lipid bilayer. To gain a better understanding of the mechanism of action, the mode of lipid bilayer spanning provides essential information. However, because of the lack of methodologies to observe it directly, it has not been revealed for the Erg-containing channel assembly for many years. In this study, we disclosed that the AmB-Erg complex spans a lipid bilayer with a single-molecule length, using solid-state nuclear magnetic resonance (NMR) experiments. Paramagnetic relaxation enhancement by Mn2+ residing near the surface of lipid bilayers induced the depth-dependent decay of 13C NMR signals for individual carbon atoms of AmB. We found that both terminal segments, the 41-COOH group and C38-C40 methyl groups, come close to the lipid bilayer surfaces, suggesting that the AmB-Erg complex spans a palmitoyloleoylphosphatidylcholine (POPC) bilayer with a single-molecule length. Molecular dynamics simulation experiments further confirmed the stabilization of the AmB-Erg complex as a single-length spanning complex. These results provide experimental evidence of the single-length complex incorporated in the membrane by making thinner a POPC-Erg bilayer that mimics fungal membranes.

Hemoglobin Value Is the Most Important Factor in the Development of Hand-Foot Syndrome under the Capecitabine Regimen.

BACKGROUND: Hand-foot syndrome (HFS) is a common side effect that has a high occurrence rate with capecitabine (Cape) chemotherapy. However, little is known about the risk factors of developing HFS under the Cape regimen. Our aim was to examine these risk factors. METHODS: A univariate analysis was used to determine the risk factors associated with developing HFS, and we calculated the effect sizes between the patients who developed HFS compared to those who did not. RESULTS: Of the 52 patients enrolled in our research, 24 (46.2%) developed HFS. This group was significantly associated with hemoglobin (Hb) values (p < 0.001), and the effect size (1.21) was more than moderate. The receiver operating characteristic curve analysis confirmed 12 mg/dl Hb as the best diagnostic cut-off value for developing HFS. The sensitivity and specificity were 75.5 and 88.2%, respectively. Patients who had Hb values of 12 or below who developed HFS had longer median times without HFS compared to patients with high Hb values (115 vs. 75 days, p = 0.30, hazard ratio = 1.42, 95% CI 0.73-2.76) and a greater area under the Kaplan-Meier curves (p < 0.05). CONCLUSION: This research suggests that the Hb value is an important factor for developing HFS.

Retrospective Analysis of the Risk Factors for Grade IV Neutropenia in Oesophageal Cancer Patients Treated with a Docetaxel, Cisplatin, and 5-Fluorouracil Regimen.

BACKGROUND: Previous Japanese trials of the docetaxel, cisplatin, and 5-fluorouracil regimen for oesophageal cancer have demonstrated that a large proportion of patients also develop grade IV neutropenia. Our aim was to examine the risk factors for neutropenia in patients treated with this regimen. METHODS: We retrospectively analysed the risk factors for developing grade IV neutropenia in 66 patients with oesophageal cancer using a multivariate analysis. RESULTS: After administering the docetaxel, cisplatin, and 5-fluorouracil regimen, 49 patients (74.2%) developed grade IV neutropenia. Grade IV neutropenia was significantly associated with platelet count (p < 0.01), alanine transaminase level (p = 0.05), and proton-pump inhibitor administration (p < 0.05). Receiver operating characteristic curve analysis confirmed a platelet count of 290 × 103/µL as the optimal diagnostic cut-off value for grade IV neutropenia. The receiver operating characteristic area for grade IV neutropenia was increased by including patients that were administered a proton-pump inhibitor and alanine transaminase level (updated model; sensitivity and specificity, 75.5 and 88.2%, respectively). CONCLUSIONS: Our findings suggest that a platelet count is the most significant predictor of grade IV neutropenia.

The Structure of the Bimolecular Complex between Amphotericin B and Ergosterol in Membranes Is Stabilized by Face-to-Face van der Waals Interaction with Their Rigid Cyclic Cores.

Amphotericin B (AmB) is a polyene macrolide antibiotic isolated from Streptomyces nodosus. The antifungal activity of AmB can be attributed to the formation of an ion-channel assembly in the presence of ergosterol (Erg), in which there are two different AmB-Erg orientations, parallel and antiparallel, as reported previously. In this study, to elucidate the structures of those AmB-Erg complexes based on solid-state nuclear magnetic resonance, a (19)F-labeled AmB derivative was newly prepared by a hybrid synthesis that utilized degradation products from the drug. Using the 2-(trimethylsilyl)ethoxymethyl (SEM) group as the protecting group for the carboxylic acid moiety of AmB, the fully deprotected labeled AmB compounds were obtained successfully. Then, these labeled AmBs were subjected to (13)C{(19)F} rotational-echo double-resonance (REDOR) experiments in hydrated lipid bilayers. The results indicated the coexistence of parallel and antiparallel orientations for AmB and Erg pairing, at a ratio of 7:3. A total of six distances between AmB and Erg were successfully obtained. Geometry analysis using the distance constraints derived from the REDOR experiments provided the plausible AmB-Erg complex structure for both the parallel and antiparallel interactions. The flat macrolide of AmB and the tetracyclic core of Erg closely contacted in a face-to-face manner, thus maximizing the van der Waals interaction between the two molecules. This interaction can be attributed to the coexistence of both the parallel and antiparallel orientations.

Role of polyol moiety of amphotericin B in ion channel formation and sterol selectivity in bilayer membrane.

Amphotericin B (AmB) is a polyene macrolide antibiotic widely used to treat mycotic infections. In this paper, we focus on the role of the polyol moiety of AmB in sterol selectivity using 7-oxo-AmB, 7α-OH-AmB, and 7ß-OH-AmB. The 7-OH analogs were prepared from 7-oxo-AmB. Their K(+) flux activity in liposomes showed that introduction of an additional ketone or hydroxy group on the polyol moiety reduces the original activity. Conformational analyses of these derivatives indicated that intramolecular hydrogen-bonding network possibly influenced the conformational rigidity of the macrolactone ring, and stabilized the active conformation in the membrane. Additionally, the flexible polyol leads to destabilization of the whole macrolactone ring conformation, resulting in a loss of sterol selectivity.

Comparative Efficacy and Safety of Standard and Biweekly Trifluridine/Tipiracil Regimen in Patients With Colorectal Cancer.

BACKGROUND/AIM: Trifluridine/tipiracil (FTD/TPI) is used to treat metastatic colorectal cancer (mCRC). Since the standard regimen of FTD/TPI features a complex dosing schedule and frequently results in severe hematological toxicities, a simplified regimen has emerged, in which FTD/TPI is orally administered biweekly. However, the survival benefits and potential adverse events associated with the biweekly FTD/TPI regimen have not been fully evaluated in previous reports. Therefore, in this study, the differences in efficacy and safety between the standard and biweekly FTD/TPI regimens were retrospectively investigated in patients with mCRC. PATIENTS AND METHODS: Data from 90 patients who received FTD/TPI for mCRC were extracted from the electronic medical records at the Osaka University Hospital. According to the inclusion and exclusion criteria, 85 of the 90 patients were enrolled in the study. We compared patient characteristics, overall survival (OS), progression-free survival (PFS), and adverse events between the standard (n=56) and biweekly groups (n=29). RESULTS: The biweekly group exhibited prolonged OS and PFS compared to patients in the standard group. Multivariate analysis for OS and PFS demonstrated that the biweekly regimen was the only significant factor that affected OS, and not PFS (HR=0.561, p=0.049). Kaplan-Meier analysis indicated that neutropenia (grade ≥3) in the biweekly group was significantly prolonged compared to the standard group (p=0.012). However, there were no significant differences in adverse events between the two groups (p>0.999). CONCLUSION: The biweekly FTD/TPI regimen, compared to the standard regimen, should enhance both OS and PFS in patients with mCRC without escalating any adverse event.

Soluble domains of cytochrome c-556 and Rieske iron-sulfur protein from Chlorobaculum tepidum: Crystal structures and interaction analysis.

In photosynthetic green sulfur bacteria, the electron transfer reaction from menaquinol:cytochrome c oxidoreductase to the P840 reaction center (RC) complex occurs directly without any involvement of soluble electron carrier protein(s). X-ray crystallography has determined the three-dimensional structures of the soluble domains of the CT0073 gene product and Rieske iron-sulfur protein (ISP). The former is a mono-heme cytochrome c with an α-absorption peak at 556 nm. The overall fold of the soluble domain of cytochrome c-556 (designated as cyt c-556sol) consists of four α-helices and is very similar to that of water-soluble cyt c-554 that independently functions as an electron donor to the P840 RC complex. However, the latter's remarkably long and flexible loop between the α3 and α4 helices seems to make it impossible to be a substitute for the former. The structure of the soluble domain of the Rieske ISP (Rieskesol protein) shows a typical ß-sheets-dominated fold with a small cluster-binding and a large subdomain. The architecture of the Rieskesol protein is bilobal and belongs to those of b6f-type Rieske ISPs. Nuclear magnetic resonance (NMR) measurements revealed weak non-polar but specific interaction sites on Rieskesol protein when mixed with cyt c-556sol. Therefore, menaquinol:cytochrome c oxidoreductase in green sulfur bacteria features a Rieske/cytb complex tightly associated with membrane-anchored cyt c-556.

Internal organ dose rate conversion coefficients of Japanese macaques to 134Cs,137Cs and 131I†.

The purpose of this study was to estimate the internal dose of radiation in Japanese macaques (aka Nihonzaru or snow monkey) due to the Fukushima nuclear power plant accident. Images of a male Japanese macaque weighing ~10 kg were acquired using a multi-slice computed tomography (CT) scan with a 64-row segment detector. The CT images were used to create voxel phantoms of the bones, bone marrow, brain, eyes, heart, lungs, stomach, liver, spleen, pancreas, kidneys, intestines, bladder, testes, thyroid and miscellaneous tissue. The Particle and Heavy Ion Transport System (PHITS) Monte Carlo code was used to calculate the internal exposure rate conversion factors for 134Cs, 137Cs and 131I isotopes for the created voxel phantoms with a statistical precision higher than 1%. The PHITS-calculated energy deposits were compared with those for rhesus monkeys. The results showed that the fractions of energy deposits for ß-radiation in different organs were almost identical between the two species. For γ-radiation, there was excellent agreement in the self-absorption rate with the approximate curve of the Japanese macaque, with an average deviation of 2%. The maximum deviation of 12% was for the kidney, which has two organs, so the error with the approximate curve is slightly larger due to the energy loss created between organs.

Amphotericin B assembles into seven-molecule ion channels: An NMR and molecular dynamics study.

Amphotericin B, an antifungal drug with a long history of use, forms fungicidal ion-permeable channels across cell membranes. Using solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we experimentally elucidated the three-dimensional structure of the molecular assemblies formed by this drug in membranes in the presence of the fungal sterol ergosterol. A stable assembly consisting of seven drug molecules was observed to form an ion conductive channel. The structure is somewhat similar to the upper half of the barrel-stave model proposed in the 1970s but substantially different in the number of molecules and in their arrangement. The present structure explains many previous findings, including structure-activity relationships of the drug, which will be useful for improving drug efficacy and reducing adverse effects.

Epoc group: transformable protecting group with gold(iii)-catalyzed fluorene formation.

This study presents the novel concept of a transformable protecting group, which changes its properties through structural transformation. Based on this concept, we developed a 2-(2-ethynylphenyl)-2-(5-methylfuran-2-yl)-ethoxycarbonyl (Epoc) group. The Epoc group was transformed into an Fmoc-like structure with gold(iii)-catalyzed fluorene formation and was removable under Fmoc-like mild basic conditions post-transformation even though it was originally stable under strongly basic conditions. As an application for organic synthesis, the Epoc group provides the novel orthogonality of gold(iii)-labile protecting groups in solid-phase peptide synthesis. In addition, the high turnover number of fluorene formation in aqueous media is suggestive of the applicability of the Epoc group to biological systems.

Bioorthogonal release of anticancer drugs via gold-triggered 2-alkynylbenzamide cyclization.

Metal-based uncaging of biomolecules has become an emerging approach for in vivo applications, which is largely due to the advantageous bioorthogonality of abiotic transition metals. Adding to the library of metal-cleavable protecting groups, this work introduces the 2-alkynylbenzamide (Ayba) moiety for the gold-triggered release of secondary amines under mild and physiological conditions. Studies were further performed to highlight some intrinsic benefits of the Ayba protecting group, which are (1) its amenable nature to derivatization for manipulating prodrug properties, and (2) its orthogonality with other commonly used transition metals like palladium and ruthenium. With a focus on highlighting its application for anticancer drug therapies, this study successfully shows that gold-triggered conversion of Ayba-protected prodrugs into bioactive anticancer drugs (i.e. doxorubicin, endoxifen) can proceed effectively in cell-based assays.

Unusual Two-Step Assembly of a Minimalistic Dipeptide-Based Functional Hypergelator.

Self-assembled peptide hydrogels represent the realization of peptide nanotechnology into biomedical products. There is a continuous quest to identify the simplest building blocks and optimize their critical gelation concentration (CGC). Herein, a minimalistic, de novo dipeptide, Fmoc-Lys(Fmoc)-Asp, as an hydrogelator with the lowest CGC ever reported, almost fourfold lower as compared to that of a large hexadecapeptide previously described, is reported. The dipeptide self-assembles through an unusual and unprecedented two-step process as elucidated by solid-state NMR and molecular dynamics simulation. The hydrogel is cytocompatible and supports 2D/3D cell growth. Conductive composite gels composed of Fmoc-Lys(Fmoc)-Asp and a conductive polymer exhibit excellent DNA binding. Fmoc-Lys(Fmoc)-Asp exhibits the lowest CGC and highest mechanical properties when compared to a library of dipeptide analogues, thus validating the uniqueness of the molecular design which confers useful properties for various potential applications.

Prescription patterns in patients with schizophrenia in Japan: First-quality indicator data from the survey of "Effectiveness of Guidelines for Dissemination and Education in psychiatric treatment (EGUIDE)" project.

BACKGROUND: Guideline for Pharmacological Therapy for Schizophrenia was published by the Japanese Society of Neuropsychopharmacology in 2015. "Effectiveness of Guidelines for Dissemination and Education in psychiatric treatment (EGUIDE)" project aimed to standardize medical practice using quality indicators (QIs) as indices to evaluate the quality of medical practice. In this study, we have reported the quality indicator values of prescription before the beginning of the guideline lectures in the EGUIDE project to ascertain the baseline status of treating patients with schizophrenia. METHODS: A cross-sectional, retrospective case record survey was conducted, involving 1164 patients with schizophrenia at the time of discharge. We checked all types and dosage of psychotropic drugs. RESULTS: Forty-three percent of patients had antipsychotic polypharmacy, and substantial concomitant medication was observed (antidepressants; 8%, mood stabilizers: 37%, anxiolytics or hypnotics: 68%). CONCLUSIONS: In the results obtained in this study, we plant to report changes in the effectiveness of education in the EGUIDE project near the future.

[Clinical features of paclitaxel-induced peripheral neuropathy and role of Gosya-jinki-gan].

Paclitaxel (PTX) is frequently used for a chemotherapy of breast cancer and gynecologic cancer. Besides a bone-marrow depression and hypersensitive reaction, the peripheral neuropathy is one of the serious adverse events of PTX. The mechanism of peripheral neuropathy has not been clarified, and few agents have been reported to be effective for the treatment and the prevention of that. Recently, it has been reported that Gosya-jinki-gan is useful for the PTX induced peripheral neuropathy, so we carried a retrospective study(n=82)to evaluate the effectiveness of Gosya-jinkigan with the medical records. It is suggested that peripheral neuropathy developed more rapidly in sequential administration of PTX every week than in administration in 4 weeks cycles consisting of 3 weeks on and 1 week off(5.4w vs. 9.4w). We have also found that Gosya-jinki-gan was possibly effective for the treatment and the prevention of peripheral neuropathy. Additionally Gosya-jinki-gan might be more effective for peripheral neuropathy when it is administered from the beginning of chemotherapy including PTX.

Real-time monitoring of the aggregation of Alzheimer's amyloid-ß via1H magic angle spinning NMR spectroscopy.

Proton magic-angle-spinning NMR used for real-time analysis of amyloid aggregation reveals that mechanical rotation of Aß1-40 monomers increases the rate of formation of aggregates, and that the increasing lag-time with peptide concentration suggests the formation of growth-incompetent species. EGCG's ability to shift off-pathway aggregation is also demonstrated.