Takayuki Okamoto

Synthesis of a poly(vinylpyrrolidone-co-dimethyl maleic anhydride) co-polymer and its application for renal drug targeting

We have synthesized a polymeric drug carrier, polyvinylpyrrolidone-co-dimethyl maleic anhydride [poly(VP-co-DMMAn)], for use in renal drug delivery. About 80% of the 10-kDa poly(VP-co-DMMAn) selectively accumulated in the kidneys 24 h after intravenous administration to mice. Although this accumulated poly(VP-co-DMMAn) was gradually excreted in the urine, about 40% remained in the kidneys 96 h after treatment. Poly(VP-co-DMMAn) was taken up by the renal proximal tubular epithelial cells and no cytotoxicity was noted. Higher doses did not produce toxicity in the kidneys or other tissues. In contrast, polyvinylpyrrolidone of the same molecular weight did not show any tissue-specific distribution. Poly(VP-co-DMMAn)-modified superoxide dismutase accumulated in the kidneys after intravenous administration and accelerated recovery from acute renal failure in a mouse model. In contrast, polyvinylpyrrolidone-modified superoxide dismutase and native superoxide dismutase were not as effective. Thus, poly(VP-co-DMMAn) is a useful candidate as a targeting carrier for renal drug delivery systems.

Incorporation of adult organ-derived endothelial cells into tumor blood vessel

In this study, we attempted to assess the incorporable potential of vascular endothelial cells derived from adult organ blood vessels into tumor blood vessels. Two kinds of adult organ-derived vascular endothelial cells, human aorta endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC), were administered into murine tumors inoculated to SCID mice. Many human blood vessel networks were visualized in the murine tumors. These cells in solid tumor not only survived and proliferated, but also incorporated into tumor endothelium. These results suggest that adult organ-derived vascular endothelial cells possess the potential to form the neovascular network in various tissues such as vascular endothelial progenitor-like cells in vivo. We propose that these cells can be regarded as a congenic (autologous) vector for vascular regeneration cell therapy and tumor vascular targeting gene therapy.

A novel cytomedical vehicle capable of protecting cells against complement

We have developed Cytomedicine, which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system. However, microencapsulated cells were not protected from humoral immune system. Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules. The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel. Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum. Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose. Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.

Development of a novel cytomedical treatment that can protect entrapped cells from host humoral immunity.

Cell therapy is expected to relieve the shortage of donors needed for organ transplantation. When patients are treated with allogeneic or xenogeneic cells, it is necessary to develop a means by which to isolate administered cells from an immune attack by the host. We have developed “cytomedicine, ” which consists of functional cells entrapped in semipermeable polymer, and previously reported that alginate-poly-l-lysine-alginate microcapsules and agarose microbeads could protect the entrapped cells from injury by cellular immunity. However, their ability to isolate from humoral immunity was insufficient. It is well known that the complement system plays an essential role in rejection of transplanted cells by host humoral immunity. Therefore, the goal of the present study was to develop a novel cytomedical device containing a polymer capable of inactivating complement. In the screening of various polymers, polyvinyl sulfate (PVS) exhibited high anticomplement activity and low cytotoxicity. Murine pancreatic β-cell line (MIN6 cell) entrapped in agarose microbeads containing PVS maintained viability and physiological insulin secretion, replying in response to glucose concentration, and resisted rabbit antisera in vitro. PVS inhibited hemolysis of sensitized sheep erythrocytes (EAs) and rabbit erythrocytes by the complement system. This result suggests that PVS inhibits both the classical and alternative complement pathways of the complement system. Next, the manner in which PVS exerts its effects on complement components was examined. PVS was found to inhibit generation of C4a and Ba generation in activation of the classical and alternative pathways, respectively. Moreover, when the EAC1 cells, which were carrying C1 on the EAs, treated with PVS were exposed to C1-deficient serum, hemolysis decreased in a PVS dose-dependent manner. These results suggest that PVS inhibits C1 in the classical pathway and C3 convertase formation in the alternative pathway. Therefore, PVS may be a useful polymer for developing an anticomplement device for cytomedical therapy.

Effects of Heparin-Urokinase, Diazepam, or Nimodipine on Brain Damage Induced by Complete Global Brain Ischemia

The outcome of brain insults induced by ischemia is influenced by two major factors, recirculation disturbance and metabolic derangement. Besides the reactive hyperemia, two types of recirculation disturbance can be distinguished; the no-reflow phenomenon [1] and delayed post-ischemic hypoperfusion [2–5], those have been shown to add a secondary ischemic insult to the tissue. Excessive release of excitatory neurotransmitters (e.g., glutamate, aspartate) during ischemia has been shown to play an important role in metabolic derangement, resulting in selective hyperexcitability after ischemia which leads to postsynaptic ionic influxes (e.g., sodium, calcium), causing neuronal damage [6–8].