Elevated expression of interleukin-6 (IL-6) and serum amyloid A (SAA) in the skin and the serum of recessive dystrophic epidermolysis bullosa: Skin as a possible source of IL-6 through Toll-like receptor ligands and SAA.

The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. In vitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.

Topical application of activator protein-1 inhibitor T-5224 suppresses inflammation and improves skin barrier function in a murine atopic dermatitis-like dermatitis.

BACKGROUND: Selective activator protein (AP)-1 inhibitors are potentially promising therapeutic agents for atopic dermatitis (AD) because AP-1 is an important regulator of skin inflammation. However, few studies have investigated the effect of topical application of AP-1 inhibitors in treating inflammatory skin disorders. METHODS: Immunohistochemistry was conducted to detect phosphorylated AP-1/c-Jun expression of skin lesions in AD patients. In the in vivo study, 1 % T-5224 ointment was topically applied for 8 days to the ears of 2,4 dinitrofluorobenzene challenged AD-like dermatitis model mice. Baricitinib, a conventional therapeutic agent Janus kinase (JAK) inhibitor, was also topically applied. In the in vitro study, human epidermal keratinocytes were treated with T-5224 and stimulated with AD-related cytokines. RESULTS: AP-1/c-Jun was phosphorylated at skin lesions in AD patients. In vivo, topical T-5224 application inhibited ear swelling (P < 0.001), restored filaggrin (Flg) expression (P < 0.01), and generally suppressed immune-related pathways. T-5224 significantly suppressed Il17a and l17f expression, whereas baricitinib did not. Baricitinib suppressed Il4, Il19, Il33 and Ifnb expression, whereas T-5224 did not. Il1a, Il1b, Il23a, Ifna, S100a8, and S100a9 expression was cooperatively downregulated following the combined use of T-5224 and baricitinib. In vitro, T-5224 restored the expression of FLG and loricrin (LOR) (P < 0.05) and suppressed IL33 expression (P < 0.05) without affecting cell viability and cytotoxicity. CONCLUSIONS: Topical T-5224 ameliorates clinical manifestations of AD-like dermatitis in mice. The effect of this inhibitor is amplified via combined use with JAK inhibitors.

Control strategy and methods for continuous direct compression processes.

We presented a control strategy for tablet manufacturing processes based on continuous direct compression. The work was conducted by the experts of pharmaceutical companies, machine suppliers, academia, and regulatory authority in Japan. Among different items in the process, the component ratio and blended powder content were selected as the items requiring the control method specific to continuous manufacturing different from the conventional batch manufacturing. The control and management of the Loss in Weight (LIW) feeder were deemed the most important, and the Residence Time Distribution (RTD) model were regarded effective for setting the control range and for controlling of the LIW feeder. Based on these ideas, the concept of process control using RTD was summarized. The presented contents can serve as a solid fundament for adopting a new control method of continuous direct compression processes in and beyond the Japanese market.

Approach to Establishment of Control Strategy for Oral Solid Dosage Forms Using Continuous Manufacturing.

As a result of the research activities of the Japan Agency for Medical Research and Development (AMED), this document aims to show an approach to establishing control strategy for continuous manufacturing of oral solid dosage forms. The methods of drug development, technology transfer, process control, and quality control used in the current commercial batch manufacturing would be effective also in continuous manufacturing, while there are differences in the process development using continuous manufacturing and batch manufacturing. This document introduces an example of the way of thinking for establishing a control strategy for continuous manufacturing processes.

Sofosbuvir/Ribavirin therapy for patients experiencing failure of ombitasvir/paritaprevir/ritonavir + ribavirin therapy: Two cases report and review of literature.

BACKGROUND: The effectiveness of sofosbuvir/ribavirin (SOF/RBV) combination therapy, which is one of the 1st-choice therapeutic options for patients with hepatitis C virus (HCV) genotype 2 (HCV-G2) in Japan according to the most recent version of the Japan Society of Hepatology guideline, for patients who experienced failure of the ombitasvir/paritaprevir/ritonavir plus ribavirin (OBV/PTV/r+RBV) combination therapy, which was another option for patients with HCV-G2, is unknown. CASE SUMMARY: We evaluated the effects of SOF/RBV combination therapy in two patients with genotype 2a who could not achieve a sustained virological response (SVR) by OBV/PTV/r+RBV combination therapy. One patient was complicated with Vogt-Koyanagi-Harada (VKH) disease. Resistance-associated variations before SOF/RBV combination therapy were not detected in two patients. Both patients had an SVR at 12 wk after the treatment (SVR12). Regarding adverse events (AEs), itching, chill, a dull feeling in the throat and cough as well as increase of alanine transaminase level were shown in one patient, while a headache and deterioration of light aversion probably due to the recurrence of VKH disease were shown in the other patients. In addition, the latter patient developed arthralgia and morning stiffness approximately 7 wk after the therapy and turned out to be diagnosed with rheumatoid arthralgia. CONCLUSION: SOF/RBV therapy might be effective for patients experiencing failure of OBV/PTV/r+RBV therapy, but caution should be taken regarding the AEs.

Global Regulatory Landscape.

Continuous manufacturing (CM) of pharmaceuticals is a rapidly growing approach in the production of active pharmaceutical ingredients and finished products. The European Medicines Agency, the US Food and Drug Administration, and the Pharmaceuticals and Medical Devices Agency have independently stated their support for the introduction of CM and provided opportunities for early dialog between industries and regulatory agencies. This paper describes the current regulatory landscape and the regulatory harmonization.

Regulatory Perspectives on Continuous Pharmaceutical Manufacturing: Moving From Theory to Practice: September 26-27, 2016, International Symposium on the Continuous Manufacturing of Pharmaceuticals.

Continuous manufacturing plays a key role in enabling the modernization of pharmaceutical manufacturing. The fate of this emerging technology will rely, in large part, on the regulatory implementation of this novel technology. This paper, which is based on the 2nd International Symposium on the Continuous Manufacturing of Pharmaceuticals, describes not only the advances that have taken place since the first International Symposium on Continuous Manufacturing of Pharmaceuticals in 2014, but the regulatory landscape that exists today. Key regulatory concepts including quality risk management, batch definition, control strategy, process monitoring and control, real-time release testing, data processing and management, and process validation/verification are outlined. Support from regulatory agencies, particularly in the form of the harmonization of regulatory expectations, will be crucial to the successful implementation of continuous manufacturing. Collaborative efforts, among academia, industry, and regulatory agencies, are the optimal solution for ensuring a solid future for this promising manufacturing technology.

General considerations regarding the in vitro and in vivo properties of block copolymer micelle products and their evaluation.

Block copolymer micelles are nanoparticles formed from block copolymers that comprise a hydrophilic polymer such as poly(ethylene glycol) and a poorly soluble polymer such as poly(amino acids). The design of block copolymer micelles is intended to regulate the in vivo pharmacokinetics, stability, and distribution profiles of an entrapped or block copolymer-linked active substance. Several block copolymer micelle products are currently undergoing clinical development; however, a major challenge in the development and evaluation of such products is identification of the physicochemical properties that affect the properties of the drug product in vivo. Here we review the overall in vitro and in vivo characteristics of block copolymer micelle products with a focus on the products currently under clinical investigation. We present examples of methods suitable for the evaluation of the physicochemical properties, non-clinical pharmacokinetics, and safety of block copolymer micelle products.

The Chlamydomonas hatching enzyme, sporangin, is expressed in specific phases of the cell cycle and is localized to the flagella of daughter cells within the sporangial cell wall.

The timely breakdown of the extracellular matrix by proteolytic enzymes is essential for development, morphogenesis and cell proliferation in plant and animal cells. Sporangin of the unicellular green alga Chlamydomonas reinhardtii that mediates breakdown of the sporangial cell wall to liberate the daughter cells after cell division is characterized as a subtilase-like serine protease. The sporangin gene is specifically transcribed during S/M phase in a synchronized vegetative cell cycle. In immunoblot analyses using a polyclonal antibody raised against the sporangin polypeptide, the enzyme is synthesized after mitotic cell division and accumulated in the daughter cells before hatching. Immunofluorescence analyses showed that sporangin is localized to the flagella of the daughter cells within the sporangial cell wall, and released into the culture medium. The data suggest that sporangin is released from flagella concurrently with the digestion of sporangial cell wall, and then the daughter cells are hatched from the sporangia in the Chlamydomonas vegetative cell cycle.

Characterization of novel genes induced by sexual adhesion and gamete fusion and of their transcriptional regulation in Chlamydomonas reinhardtii.

When mating type plus and minus gametes of Chlamydomonas are mixed, they agglutinate with each other via their flagella, fuse, then initiate the zygote formation program which includes synthesis of the zygote cell wall, fusion of nuclei and chloroplasts, and the digestion of chloroplast DNA from the minus parent. The mRNAs from gamete and zygote cells was isolated and hybridized to cDNA-macroarray filters both to identify new genes expressed during the mating reaction and the early zygote formation process and to analyze the gene expression programs that underlie these sexual processes. Twenty-one novel genes were identified in this screen, designated as EZY (early zygote expressed) genes. The EZY genes included genes encoding proteins whose function is unknown, and genes encoding proteins that appear to be involved in processes such as cell wall synthesis, gene expression, intracellular trafficking or secretion, and vesicular transport in zygotic cells. All of the EZY genes were strongly induced within 1 h during the mating process, including early zygote formation. The transcriptional characteristics of EZY genes were analyzed by using the fusion-defective mutant fus mt(+). Among the EZY genes, 12 genes were not activated in fusion-defective conditions, suggesting that cell fusion is required for their expression. The remaining nine that were transcribed in fusion-defective fus matings were also inducible by cell wall removal in either vegetative or gametic cells, indicating that these genes were induced only indirectly by the cAMP signaling pathway initiated by flagellar agglutination as a result of mating-induced cell wall loss.

Properties of human Cav2.1 channel with a spinocerebellar ataxia type 6 mutation expressed in Purkinje cells.

Spinocerebellar ataxia type 6 (SCA6) is caused by polyglutamine expansion in P/Q-type Ca2+ channels (Ca(v)2.1) and is characterized by predominant degeneration of cerebellar Purkinje cells. To characterize the Ca(v)2.1 channel with an SCA6 mutation in cerebellar Purkinje cells, we have generated knock-in mouse models that express human Ca(v)2.1 with 28 polyglutamine repeats (disease range) and with 13 polyglutamine repeats (normal range). Patch-clamp recordings of the Purkinje cells from homozygous control or SCA6 knock-in mice revealed a non-inactivating current that is highly sensitive to a spider toxin omega-Agatoxin IVA, indicating that the human Ca(v)2.1 expressed in Purkinje cells exhibits typical P-type properties in contrast to the previous data showing Q-type properties, when it was expressed in cultured cell lines. Furthermore, the voltage dependence of activation and inactivation and current density were not different between SCA6 and control, though these properties were altered in previous reports using non-neuronal cells as expression systems. Therefore, our results do not support the notion that the alteration of the channel properties may underlie the pathogenic mechanism of SCA6.

The regulatory networks of gene expression during the sexual differentiation of Chlamydomonas reinhardtii, as analyzed by mutants for gametogenesis.

Cells of Chlamydomonas reinhardtii differentiate into gametes under conditions of nitrogen (N) starvation, expressing the genes for the N-adaptation program and the gamete program. To investigate the regulatory networks of transcription among the N-starvation-inducible genes, we examined the gene expression in dif mutants, affecting gametic differentiation. In a conditional mutant, dif2, the cells remained 'vegetative' at the restrictive temperature, and the induction of 20 out of 21 genes related to the two programs was impaired. They were expressed soon after transfer of the cells to the permissive temperature, in parallel with the acquisition of mating ability. In an unconditional mutant, dif3, the cells could not differentiate into gametes at all, but the induction of only four genes (FUS1, NSG3, NSG6 and NSG7) related to the gamete program was impaired. The results suggest that Dif3 regulates putative N-starvation signal transduction pathways downstream of a master regulator, Dif2. We also examined a light-dependent laboratory strain that was unable to become gametes in the dark. The 'pre-gametes' placed in the dark, however, could induce normally all of the 21 genes, suggesting that light is required for the gametic differentiation at the translational and/or post-translational levels.

Effects of ablation of N- and R-type Ca(2+) channels on pain transmission.

Recently several mutant mouse lines lacking neuronal voltage-dependent Ca(2+) channels (VDCCs) have been established by the use of gene targeting in embryonic stem cells. Pain-related behaviors in Ca(v)2.2 (alpha(1B)) and Ca(v)2.3 (alpha(1E)) knockout mice were studied to gain further insight into the mechanism of pain transmission, where VDCCs are thought to play important roles. We review here the data from these recent studies. Ca(v)2.3-/- mice showed normal responses to acute painful stimuli, and reduced responses to the somatic inflammatory pain stimuli. Ca(v)2.3+/- mice exhibited reduced symptoms of visceral inflammatory pain. Ca(v)2.3-/- mice showed abnormal behavior related to the descending antinociceptive mechanism activated by the intraperitoneal injection of acetic acid. Ca(v)2.2-/- mice showed variable acute nociceptive responses depending on the mutant lines. However, all the lines of Ca(v)2.2-/- mice exhibited reduced responses in the phase 2 of the formalin test, suggesting a suppression of inflammatory pain. Furthermore Ca(v)2.2-/- mice showed markedly reduced neuropathic pain symptoms after spinal nerve ligation. Impaired antinociception, similar to that seen in the Ca(v)2.3-/- mice, was also observed in the Ca(v)2.2-/- mice. Therefore, it is suggested that these mutant mice could provide novel models to delineate the nociceptive and antinociceptive mechanisms.

Genealogical relationships among laboratory strains of Chlamydomonas reinhardtii as inferred from matrix metalloprotease genes.

Almost all research on Chlamydomonas reinhardtii has utilized wild-type strains of three principal stock lines (Sager, Cambridge, Ebersold-Levine), which traditionally has been assumed to be descendants of a single zygote isolated by Smith. We previously noticed that there are several sequence differences in a single-copy gene of gametolysin, mmp1, between the mt+ and mt- strains employed. To further examine the polymorphisms among the three lines, we obtained 18 representative strains of all three descendant lines of Smith's isolate, nine strains recently isolated from the wild and one strain (CC-1373 mt+) of C. smithii, a strain from Smith's collection interfertile with these C. reinhardtii strains; and we compared the mmp1-3'UTR by RFLP and sequencing analyses. Sequence divergences were found between the mt+ and mt- strains of both the Sager and Cambridge lines, but not between the two mating-type strains of the Ebersold-Levine line. We also examined the polymorphisms, using the 3'UTRs of two other mmp genes and the introns of ypt4 and fus1. Based on the results, we conclude that it is genetically impossible for all the current C. reinhardtii lines to be the immediate descendants of a single zygote.

[Structure and function of T-type calcium channels].

Molecular structure of the T-type Ca(2+) channel was first clarified by the cloning of Ca(V)3.1 (alpha(1G)) as an authentic component of the T-type channel. Since then, much progress has been made in understanding functions of the T-type Ca(2+) channels, because it has become possible to study structure-function relationship for the T-type channel using mutagenesis techniques and to examine abnormal phenotypes of T-type channel knock-out mice. Here we will review briefly the recent progress in studies on structure and function of T-type Ca(2+) channels.