Quality, safety and efficacy of follow-on biologics in Japan

Recently, WHO, EU, Japan and Canada have published guidelines on biosimilar/follow-on biologics. While there seems to be no significant difference in the general concept in these guidelines, the data to be submitted for product approval are partially different. Differences have been noted in the requirements for comparability studies on stability, prerequisites for reference product, or for the need of comparability exercise for determination of process-related impurities. In Japan, there have been many discussions about the amount and extent of data for approval of follow-on biologics. We try to clarify the scientific background and rational for regulatory pathway of biosimilar/follow-on biologics in Japan in comparison with the guidelines available from WHO, EU and Canada. In this article, we address and discuss the scientific background underlying these differences to facilitate the harmonization of follow-on biologic principles in the guidelines in future.     

Gene transfer using micellar nanovectors inhibits choroidal neovascularization in vivo

Purpose

Age-related macular degeneration caused by choroidal neovascularization (CNV) remains difficult to be treated despite the recent advent of several treatment options. In this study, we investigated the in vivo angiogenic control by intravenous injection of polyion complex (PIC) micelle encapsulating plasmid DNA (pDNA) using a mice CNV model.

Methods

The transfection efficiency of the PIC micelle was investigated using the laser-induced CNV in eight-week-old male C57 BJ/6 mice. Firstly, each mouse received intravenous injection of micelle encapsulating pDNA of Yellow Fluorescent Protein (pYFP) on days 1,3 and 5. The expression of YFP was analyzed using fluorescein microscopy and western blotting analysis. In the next experiments, each mouse received intravenous injection of micelle encapsulating pDNA of soluble Fms-like tyrosine kinase-1 (psFlt-1) 1,3 and 5 days after the induction of CNV and the CNV lesion was analyzed by choroidal flatmounts on day 7.

Results

Fluorescein microscopy and western blotting analysis revealed that the expression of YFP was confirmed in the CNV area after injection of the PIC micelle, but the expression was not detected neither in mice that received naked pDNA nor those without CNV. Furthermore, the CNV area in the mice that received intravenous injection of the psFlt-1-encapsulated PIC micelle was significantly reduced by 65% compared to that in control mice (p<0.01).

Conclusions

Transfection of sFlt-1 with the PIC micelle by intravenous injection to mice CNV models showed significant inhibition of CNV. The current results revealed the significant potential of nonviral gene therapy for regulation of CNV using the PIC micelle encapsulating pDNA.     

Tight junction regulates epidermal calcium ion gradient and differentiation

It is well known that calcium ions (Ca²⁺) induce keratinocyte differentiation. Ca²⁺ distributes to form a vertical gradient that peaks at the stratum granulosum. It is thought that the stratum corneum (SC) forms the Ca²⁺ gradient since it is considered the only permeability barrier in the skin. However, the epidermal tight junction (TJ) in the granulosum has recently been suggested to restrict molecular movement to assist the SC as a secondary barrier. The objective of this study was to clarify the contribution of the TJ to Ca²⁺ gradient and epidermal differentiation in reconstructed human epidermis. When the epidermal TJ barrier was disrupted by sodium caprate treatment, Ca²⁺ flux increased and the gradient changed in ion-capture cytochemistry images. Alterations of ultrastructures and proliferation/differentiation markers revealed that both hyperproliferation and precocious differentiation occurred regionally in the epidermis. These results suggest that the TJ plays a crucial role in maintaining epidermal homeostasis by controlling the Ca²⁺ gradient.     

Purification and characterization of tributyltin-binding protein type 2 from plasma of Japanese flounder, Paralichthys olivaceus

We used gel filtration chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis to purify tributyltin-binding protein type 2 (TBT-bp 2) from plasma of Japanese flounder (Paralichthys olivaceus) injected intraperitoneally with TBT (5.0 mg/kg body weight). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the molecular mass of TBT-bp 2 was approximately 48 kDa, and isoelectric focusing-polyacrylamide gel electrophoresis indicated that the isoelectric point was approximately 3.0. TBT-bp 2 contained 40% N-glycan. The complete cDNA nucleotide sequence and the genome sequence of TBT-bp 2 were determined by means of rapid amplification of cDNA ends of liver tissue of Japanese flounder and a genome-walking technique, respectively. The 216 amino acid sequence of TBT-bp 2 showed 47% identity to the sequences of puffer fish (Takifugu pardalis) saxitoxin- and tetrodotoxin-binding protein but only 27% similarity to the sequence of TBT-bp 1. Analysis of the motif sequence of the amino acid sequence and the structure of the gene encoding TBT-bp 2 suggested that this protein belongs to the lipocalin superfamily.     

The evolutionary process of bioluminescence and aposematism in cantharoid beetles (Coleoptera: Elateroidea) inferred by the analysis of 18S ribosomal DNA

Cantharoid beetles are distinctive for their leathery soft elytra and conspicuous color or bioluminescence, and many of the members are equipped with chemical defenses. Thus, the vivid coloration of Cantharidae and Lycidae and the bioluminescence in Lampyridae and Phengodidae appear to be aposematic signals. However, the evolutionary aspect of their aposematism is not well understood, because the classification of the families remains controversial. In this study, we performed molecular phylogenetic analyses of species from cantharoid families, based on nucleotide sequence comparisons of nuclear 18S ribosomal DNA. The results shows that the luminous species Rhagophthalmus ohbai, which had sometimes been classified in Lampyridae, is excluded from a lampyrid clade and associates with the taxa of Phengodidae. The molecular data also suggests that four major subfamilies of Cantharidae (Cantharinae, Chauliognathinae, Malthininae, and Silinae) form a clade. The six subfamilies of Lampyridae are grouped and classified into two sublineages: Amydetinae + Lampyrinae + Photurinae and Cyphonocerinae + Luciolinae +Ototretinae. Genera Drilaster and Stenocladius are the members of Ototretinae in Lampyridae. These results conform to traditional taxonomy but disagree with more recent cladistic analyses. Based on these findings, we propose an evolutionary process of bioluminescence and aposematism in cantharoids: the clades of Cantharidae, Lampyridae, Lycidae, and Phengodidae have evolved aposematic coloration; subsequently Lampyridae and Phengodidae acquired bioluminescence; and these four major cantharoid families achieved their current adaptive diversities.     

Firefly luciferase genes from the subfamilies Psilocladinae and Ototretinae (Lampyridae, Coleoptera)

Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae (Cyphonocerus ruficollis) and Ototretinae (Drilaster axillaris and Stenocladius azumai), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81-89% identities to each other and 60-81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545-546 nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550-568 nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.     

Characterization of overall ceramide species in human stratum corneum

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of alpha-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C(18))-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.     

The effect of season on serum testosterone concentrations in dogs

Photoperiod and environmental temperature are two important factors that may influence the reproductive cycle of various species. The objective of this study was to investigate seasonal influences on serum testosterone concentrations in dogs in a tropical zone, where the variation in day length between winter and summer solstice was approximately 2.5 h. Blood samples were collected every 15 days from seven adult dogs over a 14-month interval and serum testosterone concentrations were determined by radioimmunoassay. The year was divided into four seasons and mean testosterone concentrations for each season were related to the mean environmental temperature and rainfall during that season. Mean testosterone concentrations were 1.81 ng/mL (winter 2002), 1.93 ng/mL (spring 2002), 1.31 ng/mL (summer 2003), 2.02 ng/mL (autumn 2003) and 1.93 ng/mL (winter 2003). The temperature ranged from 10.2 to 32.8 degrees C and the rainfall from 33 to 476 mm. Serum testosterone concentrations were lower in summer 2003 than in both spring 2002 (P = 0.05) and autumn 2003 (P = 0.016). In a tropical zone, a combination of high temperature and substantial rainfall may have reduced serum testosterone concentrations in dogs.