pH-Sensitive Amphiphilic Diblock Polyphosphoesters with Lactate Units: Synthesis and Application as Drug Carriers.

pH-sensitive amphiphilic diblock polyphosphoesters containing lactic acid units were synthesized by multistep one-pot polycondensation reactions. They comprise acid-labile P(O)-O-C and C(O)-O-C bonds, the cleavage of which depends on the pH of the medium. The structure of these copolymers was characterized by 1H, 13C {H}, 31P NMR, and size exclusion chromatography (SEC). The newly synthesized polymers self-assembled into the micellar structure in an aqueous solution. The effects of the molecular weight of the copolymer and the length of the hydrophobic chain on micelle formation and stabilityand micelle size were studied via dynamic light scattering (DLS). Drug loading and encapsulation efficiency tests using doxorubicin revealed that hydrophobic drugs can be delivered by copolymers. It was established that the molecular weight of the copolymer, length of the hydrophobic chain and content of lactate units affects the size of the micelles, drug loading, and efficiency of encapsulation. A copolymer with 10.7% lactate content has drug loading (3.2 ± 0.3) and efficiency of encapsulation (57.4 ± 3.2), compared to the same copolymer with 41.8% lactate content (1.63%) and (45.8%), respectively. It was demonstrated that the poly[alkylpoly(ethylene glycol) phosphate-b-alkylpoly(ethylene glycol)lactate phosphate] DOX system has a pH-sensitive response capability in the result in which DOX was selectively accumulated into the tumor, where pH is acidic. The results obtained indicate that amphiphilic diblock polyphosphoesters have potential as drug carriers.

Phosphoric Acid Triester Micelles: Characterization and Self-Assembly.

In this study, we analyzed the properties of amphiphilic alkyldi(methoxy poly(ethylene glycol) (MePEG)350-lactate) phosphates based on ethyl lactate, the monomethyl ether of poly(ethylene glycol)350, and alkyldichloro phosphates. Interestingly, these triesters combine two biodegradable bonds, -P(O)-O-C and -C(O)-O-C-, and include hydrophilic (MePEG350-lactate) and hydrophobic (R-aliphatic chain of alcohols) moieties. The properties of these esters resemble those of phospholipids. After being placed in an aqueous solution, they self-assembled. We also determined the effects of ester composition on micelle formation, stability, and size using dynamic light scattering. Solubilization tests using Sudan III or doxorubicin hydrochloride (Dox·HCl) revealed that they could be incorporated into the hydrophobic cores of dodecyl di(MePEG350-lactate) phosphate and hexadecyl di(MePEG350-lactate) phosphate. Notably, dodecyl di(MePEG350-lactate) phosphate was stable for five days, whereas hexadecyl di(MePEG350-lactate) phosphate was stable for seven days in phosphate-buffered saline. Moreover, Dox·HCl release rates from the micelles were approximately 30-40, 70-80, and 90-100% after 1, 5, and 28 d, respectively.

Synthesis and Characterization of Amphiphilic Diblock Polyphosphoesters Containing Lactic Acid Units for Potential Drug Delivery Applications.

Multistep one-pot polycondensation reactions synthesized amphiphilic diblock polyphosphoesters containing lactic acid units in the polymer backbone. At the first step was synthesized poly[poly(ethylene glycol) H-phosphonate-b-poly(ethylene glycol)lactate H-phosphonate] was converted through one pot oxidation into poly[alkylpoly(ethylene glycol) phosphate-b-alkylpoly(ethylene glycol)lactate phosphate]s. They were characterized by 1H, 13C {H},31P NMR, and size exclusion chromatography (SEC). The effects of the polymer composition on micelle formation and stability, and micelle size were studied via dynamic light scattering (DLS). The hydrophilic/hydrophobic balance of these polymers can be controlled by changing the chain lengths of hydrophobic alcohols. Drug loading and encapsulation efficiency tests using Sudan III and doxorubicin revealed that hydrophobic substances can be incorporated inside the hydrophobic core of polymer micelles. The micelle size was 72-108 nm when encapsulating Sudan III and 89-116 nm when encapsulating doxorubicin. Loading capacity and encapsulation efficiency depend on the length of alkyl side chains. Changing the alkyl side chain from 8 to 16 carbon atoms increased micelle-encapsulated Sudan III and doxorubicin by 1.6- and 1.1-fold, respectively. The results obtained indicate that these diblock copolymers have the potential as drug carriers.

Thermoresponsive Polyphosphoester via Polycondensation Reactions: Synthesis, Characterization, and Self-Assembly.

Using a novel strategy, amphiphilic polyphosphoesters based on poly(oxyethylene H-phosphonate)s (POEHP) with different poly(ethylene glycol) segment lengths and aliphatic alcohols with various alkyl chain lengths were synthesized using polycondensation reactions. They were characterized by 1H NMR, 13C {H} NMR 31P NMR, IR, and size exclusion chromatography (SEC). The effects of the polymer structure on micelle formation and stability, micelle size, and critical micelle temperature were studied via dynamic light scattering (DLS). The hydrophilic/hydrophobic balance of these polymers can be controlled by changing the chain lengths of hydrophilic PEG and hydrophobic alcohols. A solubilizing test, using Sudan III, revealed that hydrophobic substances can be incorporated inside the hydrophobic core of polymer associates. Loading capacity depends on the length of alkyl side chains. The results obtained indicate that these structurally flexible polymers have the potential as drug carriers.

Enthalpy-Entropy Compensation in the Structure-Dependent Effect of Nonsteroidal Anti-inflammatory Drugs on the Aqueous Solubility of Diltiazem.

Certain combinations of acidic and basic drugs can cause significant changes in physicochemical properties through the formation of ionic liquids, eutectic mixtures, or deep eutectic solvents. In particular, combining indomethacin and lidocaine is known to result in apparent increases in both the partition coefficients (hydrophobicity) and aqueous solubilities (hydrophilicity). The physicochemical interactions between drugs change the water solubility of the drugs and affect the bio-availability of active pharmaceutical ingredients. Therefore, we need to clarify the mechanism of changes of water solubility of drugs through the physicochemical interactions. In the present study, we identified a thermodynamic factor that regulates the dissolution of a basic drug, in the presence of various acidic nonsteroidal anti-inflammatory drugs. The results demonstrated that enthalpy-entropy compensation plays a key role in the dissolution of drug mixtures and that relevant thermodynamic conditions should be considered.

Discontinuation and remission rates and social functioning in patients with schizophrenia receiving second-generation antipsychotics: 52-week evaluation of JUMPs, a randomized, open-label study.

AIM: Globally, evidence from short-term studies is insufficient for the guidelines to uniformly recommend a particular antipsychotic(s) for the maintenance treatment of schizophrenia. Therefore, long-term comprehensive evaluation of antipsychotics is required from a social rehabilitation perspective, especially for drugs that have not yet been studied. The Japan Useful Medication Program for Schizophrenia (JUMPs) is a large-scale, long-term naturalistic study to present pivotal 52-week data on the continuity of second-generation antipsychotics (SGA: aripiprazole, blonanserin, and paliperidone). METHODS: JUMPs was an open-label, three-arm, randomized, parallel-group, 52-week study. Enrolled patients had schizophrenia, were ≥20 years old, and required antipsychotic treatment or switched from previous therapy. The primary endpoint was treatment discontinuation rate over 52 weeks. Secondary outcomes included remission rate, social functioning, and quality-of-life scores [Personal and Social Performance Scale (PSP) and EuroQol-5 dimensions], and safety. RESULTS: In total, 251 patients received aripiprazole (n = 82), blonanserin (n = 85), or paliperidone (n = 84). The discontinuation rate (P = 0.9771) and remission rates (P > 0.05) over 52 weeks did not differ significantly between the three treatment groups. The discontinuation rates were 68.3%, 68.2%, and 65.5% in the aripiprazole, blonanserin, and paliperidone groups, respectively. Significant improvements (all P < 0.05) from baseline in PSP scores were observed at start of monotherapy, week 26, and week 52 in the overall cohort and blonanserin group and at week 26 in the aripiprazole group. The adverse event profile favored blonanserin. CONCLUSION: All three SGAs evaluated in this study showed similar treatment discontinuation rates in patients with chronic schizophrenia in Japan.

Uniformity and Efficacy of Dry Powders Delivered to the Lungs of a Mycobacterial-Surrogate Rat Model of Tuberculosis.

PURPOSE: Pulmonary administration of dry drug powder is a considered promising strategy in the treatment of various lung diseases such as tuberculosis and is more effective than systemic medication. However, in the pre-clinical study phase, there is a lack of devices for effective delivery of dry powders to the lungs of small rodents. In this study, an administration device which utilizes Venturi effect to deliver dry powders to the lungs homogeneously was developed. METHODS: A Venturi-effect administration device which synchronizes with breathes by use of a ventilator and aerosolizes the dry powders was created. Pulmonary distribution of inhalable dry powders prepared by spray-drying poly(lactic-co-glycolic) acid and an antituberculosis agent rifampicin and anti-tuberculosis effect of the powders on mycobacteria infected rats by administration with the Venturi-effect administration device and a conventional insufflation device were evaluated. RESULTS: Homogeneous distribution of the dry powders in the lung was achieved by the Venturi-effect administration device due to efficient and recurring aerosolization of loaded dry powders while synchronizing with breathes. Amount of rifampicin delivered to the lungs by the Venturi-effect administration device was three times higher than that by a conventional insufflation device, demonstrating three times greater antimycobacterial activity. CONCLUSIONS: The Venturi-effect administration device aerosolized inhalable antituberculosis dry powders efficiently, achieved uniform pulmonary distribution, and aided the dry powders to exert antituberculosis activity on lung-residing mycobacteria.

Return-to-Work Screening by Linear Discriminant Analysis of Heart Rate Variability Indices in Depressed Subjects.

Using a linear discriminant analysis of heart rate variability (HRV) indices, the present study sought to verify the usefulness of autonomic measurement in major depressive disorder (MDD) patients by assessing the feasibility of their return to work after sick leave. When reinstatement was scheduled, patients' HRV was measured using a wearable electrocardiogram device. The outcome of the reinstatement was evaluated at one month after returning to work. HRV indices including high- and low-frequency components were calculated in three conditions within a session: initial rest, mental task, and rest after task. A linear discriminant function was made using the HRV indices of 30 MDD patients from our previous study to effectively discriminate the successful reinstatement from the unsuccessful reinstatement; this was then tested on 52 patients who participated in the present study. The discriminant function showed that the sensitivity and specificity in discriminating successful from unsuccessful returns were 95.8% and 35.7%, respectively. Sensitivity is high, indicating that normal HRV is required for a successful return, and that the discriminant analysis of HRV indices is useful for return-to-work screening in MDD patients. On the other hand, specificity is low, suggesting that other factors may also affect the outcome of reinstatement.

Assessment of Blood Plasma Free-amino Acid Levels in Infertile Men.

BACKGROUND/AIM: The objective of this study was to investigate the significance of the blood levels of free amino acids (AAs) in infertile men. PATIENTS AND METHODS: Ninety-three men who underwent examinations for infertility were included. The concentrations of 20 AAs were measured and compared in four groups (normospermia, obstructive azoospermia, oligozoospermia, non-obstructive azoospermia) based on semen analysis and clinical parameters. RESULTS: When the 93 men were divided into normospermia, obstructive azoospermia, oligozoospermia, and non-obstructive azoospermia groups, no significant differences were found in the concentrations of the 20 AAs between them. We then compared 49 men diagnosed with normozoospermia or oligozoospermia according to the median sperm motility and morphology abnormalities rates (30% and 20%, respectively). Men with low motility rates had significantly lower levels of tryptophan and alanine, and men with high abnormal morphology rates had significantly lower levels of aspartate and glutamate. CONCLUSION: AAs are probably involved in the pathogenesis of male infertility, particularly oligozoospermia.

Total Diet Study to Assess Radioactive Cs and 40K Levels in the Japanese Population before and after the Fukushima Daiichi Nuclear Power Plant Accident.

We conducted a total diet study (TDS) of 137Cs, 134Cs, and 40K to assess their average dietary exposure levels in a Japanese adult population before and after the Fukushima Daiichi nuclear power plant (FDNPP) accident. Nineteen market baskets were evaluated in 2006-2011. In each basket, a TDS sample comprising tap water and 160-170 food items, which were combined into 13 groups, were collected for analysis by gamma-ray spectrometry. From 2006 to 2010, the 137Cs activity concentration in the "fish and shellfish" group was 0.099 Bq/kg, representing the highest value obtained, whereas the total committed effective dose (CED) of radiocesium isotopes (137Cs + 134Cs) was 0.69 µSv. In 2011, "milk and dairy products" from Sendai City had a Cs activity concentration of 12 Bq/kg, representing the highest values among all food groups studied. However, the annual CED of radioactive Cs in Fukushima City was 17 µSv after the FDNPP accident, which is 60-fold lower than the maximum permissible dose of 1 mSv/year. The mean CED obtained for 40K was 180 µSv, which is comparable to the global average. Our results reveal the average dietary exposure of 137Cs, 134Cs, and 40K, which can aid in estimating the radiological safety of foods.

Molecular recognizable ion-paired complex formation between diclofenac/indomethacin and famotidine/cimetidine regulates their aqueous solubility.

This study focused on the physicochemical interactions between acidic and basic drugs in aqueous solutions. Their ion pair interactions were evaluated in an in vitro study. The model non-steroid anti-inflammatory drugs (NSAIDs), indomethacin (INM) and diclofenac (DIC), were used as acidic and hydrophobic drugs, whereas cimetidine (CIM), famotidine (FAM), and imidazole (IMD) were used as basic additives with heterocyclic moieties. The drug mixtures were evaluated by thermal analysis, dissolution test, nuclear magnetic resonance (NMR) spectroscopy, and mass spectroscopy. The fusion enthalpy of DIC-CIM, INM-CIM, and INM-arginine (ARG) sample was calculated based on melting temperature transformation. The DIC mixture with CIM, IMD, antipyrine (ANT), and ARG showed enhanced solubility, whereas the DIC-FAM mixture sample showed a decreased solubility. Electrospray ionization mass spectroscopy was carried out to detect binary mixtures. The interactions in DIC-FAM mixture sample were found between the carboxyl group of DIC and the amine groups of FAM by NMR. These findings were suggested that DIC-FAM mixture samples construct ion pair complexes based on the theory of Bjerrum. Moreover, the acid model drug and basic model drug also can be constructed 1:1 complexes that affects their solubility in the solvent of water type.

Usefulness of heart rate variability indices in assessing the risk of an unsuccessful return to work after sick leave in depressed patients.

AIM: The present study aimed to examine whether heart rate variability (HRV) indices in depressed patients measured at return to work after sick leave are related to the outcome of reinstatement. METHODS: This study included 30 workers who took a leave of absence due to major depressive disorder. HRV was measured twice, once when participants left work and another when they returned to work. One month after returning to work, 19 participants continued their original work (successful return group), while 11 failed to perform their original work (unsuccessful return group). HRV indices including high- and low-frequency components (HF and LF) were calculated in three conditions within a session lasting for about 5 minutes, initial rest (Rest), mental task (Task), and rest after task (After), and were compared between the two participant groups. Psychological states were evaluated using Self-rating Depression Scale and State-Trait Anxiety Inventory. RESULTS: No significant differences were observed in the HRV indices on leaving work between groups. On returning to work, the "unsuccessful return group" exhibited lower HF Rest score, higher HF Task/Rest ratio, and higher LF/HF Rest score than the "successful return group." Psychological scores improved in both groups. CONCLUSION: These results indicate that autonomic dysregulations revealed by HRV measurement at return to work after a leave of absence in MDD patients were related to the outcome of reinstatement and can serve as useful information for the assessment of the risk of unsuccessful return.

Mastication Affects Transcriptomes of Mouse Microglia.

BACKGROUND/AIM: We investigated whether mastication affects microglia, whose activity is thought to be associated with cognition and brain tumor progression. MATERIALS AND METHODS: We kept mice by feeding either a hard or soft diet for 2, 4 or 8 months. After each period, we removed the whole brains and isolated microglia. The total RNA extracted from each brain's microglia was subjected to DNA microarray analysis. RESULTS: Many genes were found to be significantly differentially expressed between hard- and soft-diet-fed mice in each group of the same feeding period. The expression of several genes involved in the regulation of actin cytoskeleton was down-regulated in the soft-diet-fed mice. CONCLUSION: Mastication may affect microglia's roles in cognition as well as their neuroimmune activity through their activity of patrolling the brain.

The 31-nucleotide Y4-RNA fragment in plasma is a potential novel biomarker.

The 31- and 32-nt 5'-fragment of Y4-RNA (Y4RNAfr) exists abundantly in human peripheral blood plasma. Although physiological roles of the plasma Y4RNAfr are not well established, its potential utility as a diagnostic/prognostic marker for acute coronary syndrome was suggested. In this paper, to establish a normal range of the Y4RNAfr level in plasma, we measured plasma Y4RNAfr levels of 40 healthy persons using the method we have developed, and compared them with other blood test data. From the obtained data, we tentatively regarded <0.1 fmol/ng as normal for the Y4RNAfr level in peripheral blood plasma. And the white blood cell count (WBC) and the C-reactive protein (CRP) level showed moderate positive correlations with the Y4RNAfr level, suggesting that Y4RNAfr could be a potential novel inflammatory marker. We also measured the Y4RNAfr level in peripheral blood plasma from four multiple myeloma patients. The plasma Y4RNAfr level was abnormal in all four myeloma patients, and the levels for two patients were far beyond the normal level. The WBC for each patient was normal and the CRP levels for two patients were normal. These observations together suggest that a high level of Y4RNAfr in peripheral blood plasma and a normal WBC could be indicative of multiple myeloma.

The function of oxybuprocaine: a parachute effect that sustains the supersaturated state of anhydrous piroxicam crystals.

Polymers have been recognized to have the function of sustaining the supersaturated state of drugs. This function has been widely studied because it will improve the absorption of poorly water-soluble drugs. However, clarifying the mechanism of this sustaining pharmaceutical effect (parachute effect) on the supersaturated state as a result of polymers is remains a task. We have found that oxybuprocaine, which is a small molecule, has a parachute effect on the supersaturated state (due to an anhydrate-to-hydrate transformation) of piroxicam-anhydrate in the aqueous phase. We consider that oxybuprocaine controls the environment of the solution and the network of polymers is unnecessary. Therefore, oxybuprocaine not only becomes a clue for elucidating the essential mechanism of the parachute effect of polymers but also enables us to rationally propose a new type of solubilizer.

Functional analysis of coiled-coil domains of MCU in mitochondrial calcium uptake.

The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel. This complex consists of MCU, mitochondrial calcium uptake proteins (MICUs), MCU regulator 1 (MCUR1), essential MCU regulator element (EMRE), etc. MCU, which is the pore-forming subunit, has 2 highly conserved coiled-coil domains (CC1 and CC2); however, their functional roles are unknown. The yeast expression system of mammalian MCU and EMRE enables precise reconstitution of the properties of the mammalian MCU complex in yeast mitochondria. Using the yeast expression system, we here showed that, when MCU mutant lacking CC1 or CC2 was expressed together with EMRE in yeast, their mitochondrial Ca2+-uptake function was lost. Additionally, point mutations in CC1 or CC2, which were expected to prevent the formation of the coiled coil, also disrupted the Ca2+-uptake function. Thus, it is essential for the Ca2+ uptake function of MCU that the coiled-coil structure be formed in CC1 and CC2. The loss of function of those mutated MCUs was also observed in the mitochondria of a yeast strain lacking the yeast MCUR1 homolog. Also, in the D. discoideum MCU, which has EMRE-independent Ca2+-uptake function, the deletion of either CC1 or CC2 caused the loss of function. These results indicated that the critical functions of CC1 and CC2 were independent of other regulatory subunits such as MCUR1 and EMRE, suggesting that CC1 and CC2 might be essential for pore formation by MCUs themselves. Based on the tetrameric structure of MCU, we discussed the functional roles of the coiled-coil domains of MCU.

Therapeutic Effect of GGsTop, Selective Gamma-glutamyl Transpeptidase Inhibitor, on a Mouse Model of 5-Fluorouracil-induced Oral Mucositis.

BACKGROUND: Oral mucositis (OM) induced by cancer chemotherapy has a high incidence and serious symptoms, which often force chemotherapy to be stopped. GGsTop is a newly-discovered gamma-glutamyl transpeptidase (GGT) inhibitor. Previous research suggested that inhibition of GGT suppressed reactive oxygen species and induced the production of collagen and elastin. We hypothesized that GGsTop could safely treat OM. MATERIALS AND METHODS: A mouse model of OM was treated with GGsTop and ulcer area, weight, and white blood cell count were determined. The treatment effect was also evaluated by hematoxylin-eosin and collagen staining. RESULTS: The therapeutic effect of GGsTop was better than that of an existing drug and may be safely used in combination with chemotherapy. Furthermore, GGsTop promoted collagen production in oral mucosa. CONCLUSION: GGsTop treated OM quickly and safely. GGsTop is highly valuable for use as a treatment for OM.

Transdermal delivery of 40-nm silk fibroin nanoparticles.

Transdermal administration of drugs improves their bioavailability and is capable of systemic and local treatment. To improve the skin permeability of drugs, nano-sized systems have attracted attention as drug carriers for transdermal drug delivery system. We considered that silk fibroin composed of a crystalline region with many hydrophobic amino acids and an amorphous region with many hydrophilic amino acids was useful as a carrier for transdermal administration of a drug because of the balance between hydrophilicity and hydrophobicity. In this study, silk fibroin nanoparticles with mean volume diameters of 42.3 nm were successfully prepared, and storage stability was confirmed by storing the nanoparticle suspension at 4, 32, and 37 °C for a week. At any storage temperature, the mean volume diameter and standard deviation were stable. The polydispersity indexes were 0.19-0.23, and no specific trends were observed. Then, to investigate the transdermal delivery route of the silk fibroin nanoparticles, skin permeability in vivo was evaluated using mice. Six hours after administration, fluorescent substances were observed in the dermis in addition to the stratum corneum, hair follicles and the epidermis around them. This result indicated that fibroin nanoparticles with the mean volume diameter of 40-nm penetrated the stratum corneum and was delivered deep into the skin. Therefore, it was suggested that small nanoparticles prepared using silk fibroin are useful for drug delivery to the dermis.

Polyethyleneimine renders mitochondrial membranes permeable by interacting with negatively charged phospholipids in them.

Polyethyleneimines (PEIs) are used for transfection of cells with nucleic acids. Meanwhile, the interaction of PEI with mitochondria causes cytochrome c release prior to apoptosis; the mechanisms how PEI causes this permeabilization of mitochondrial membranes and the release of cytochrome c remain unclear. To clarify these mechanisms, we examined the effects of branched-type PEI and linear-type PEI, each of which was 25 kDa in size, on mitochondria. The permeabilization potency of mitochondrial membranes by branched PEI was stronger than that by linear PEI. The permeabilization by PEIs were insensitive to permeability-transition inhibitors, indicating that PEI-induced permeabilization was not attributed to permeability transition. Meanwhile, PEIs caused permeabilization of artificial lipid vesicles; again, the permeabilization potency of branched PEI was stronger than that of linear PEI. Such a difference in this potency was close to that in the case of isolated mitochondria, signifying that the PEI-induced permeabilization of mitochondrial membranes could be attributed to PEI's interaction with the phospholipid phase. Furthermore, this PEI-induced permeabilization of the lipid vesicles was observed only in the case of lipid vesicles including negatively charged phospholipids. These results indicate that PEIs interacted with negatively charged phospholipids in the mitochondrial membranes to directly lead to their permeabilization.

Decarboxylation of indomethacin induced by heat treatment.

As crystalline indomethacin is heated and subsequently cooled, it transforms into glassy indomethacin. While the original crystals are off-white in color, the glass becomes blackish-brown via a yellow intermediate stage. TLC of the components of the glass revealed three bands. The yellow component, which is generated either under hypoxic conditions or in the dark, was elucidated by NMR spectroscopy to be a decarboxylated fragment produced by thermal degradation. The colorless component is proposed to be formed by the opening of the indole ring of indomethacin; the structure of this degradation product was identified by EI-MS to be the same as the oxidative-cleavage product formed upon UV-irradiation, as previously reported. Another band was a blackish-brown pigment whose mobility placed it close to the TLC baseline. This oxidative-cleavage product and the blackish-brown pigment are not generated under hypoxic conditions. However, the extent of indomethacin decarboxylation under hypoxic conditions was found to be dependent on the heating temperature and time. Consequently, we prepared amorphous indomethacin through control of the heating temperature and time; heating at 160 °C for 30 min or less under hypoxic conditions is optimum for obtaining pure amorphous indomethacin.