Several reconstructed cultured human skin models (RSMs) are already utilized as membrane alternatives to human and animal skin in skin corrosive/irritation tests. They are also utilized in skin permeation experiments from the viewpoint of animal welfare; however, different permeation profiles of chemicals were found between RSMs and excised human or animal skin. RSMs and excised human skin were morphologically evaluated by a light microscope and a transmission electron microscope. In the results, the micromorphology of all RSMs differed from that of human skin. In particular, the lamellar layer between corneocytes in RSMs was much narrower than that in the human stratum corneum. The lamella layer affects not only the diffusion and partition properties of chemical compounds in RSMs but also the concentration-distance profile of chemicals in the models. Furthermore, esterase distribution in RSMs was different to that in human skin. This difference would certainly affect the permeation of both parent ester compounds and their metabolites through RSMs. Evaluation of the morphological and enzymatic differences between RSMs and human skin would be helpful to understand the differences in the chemical permeation profiles between RSMs and human skin.
Appropriate preclinical angiogenic screening is a preeminent consideration for the development of agents that target the tumor microvessels coordination among endothelial cells and pericytes. We previously investigated implantation of a thermoreversible gelation polymer (TGP) disc in an in vivo rat angiogenesis model (Wakui et al., 2006), but this model required many animals and long-term experiments. To reduce and replace animal usage, the present study developed an in vitro TGP disc rat angiogenesis model using a TGP disc pretreated to induce microvessels within five days after implantation in subcutaneous tissue of the rat back. Electron microscopic analysis revealed a low number of small-sized immature capillaries after 24 hours in vitro incubation. After 48 hours incubation, a number of more developed capillaries with narrow lumens were dominantly observed. After 72 hours incubation, the capillary density had decreased, and mature and also degenerated capillaries were observed. Quantitative RT-PCR analysis revealed that the level of Ang-1 mRNA was low after 24-48 hours incubation, and it had increased at 72 hours incubation, while a high level of Ang-2 mRNA was observed at 24-72 hours incubation. In the present in vitro angiogenesis model, the microvessel growth stage was 24-48 hours and the microvessel maturation stage was after 72 hours incubation.
The liver, kidney, intestine and brain possess a wide variety of drug transporters which are very important attributes in drug disposition, safety and efficacy. Consequently, it is now well recognized that obtaining information regarding drug transporters is fundamental during novel drug development. Recent molecular cloning endeavors have identified several families (i.e. SLC22 family) of multi-specific drug transporters including organic anion and organic cation transporters (OATs/OCTs). However, genome-based drug discovery has resulted in its own set of difficulties (i.e. species differences between human and other animals) and will require animals to examine the disposition of drugs in vivo. Herein, we present an example regarding organic solute transport properties and species differences; we summarize data regarding substrate specificities among OATs/OCTs obtained from human drug transporter gene-expressing cells. The presented substrate specificity data suggest that contribution to pharmacokinetics appears to be different for each drug in vivo. Therefore, the introduction of these transporter gene-expressing cells in early drug development will lead to contributions not only in refinement, but will assist in the reduction and replacement (i.e. the three R's concept: Reduction, Replacement and Refinement) for alternatives to animal usage.