Kazuo Umemura

Adacolumn, an adsorptive carrier based granulocyte and monocyte apheresis device for the treatment of inflammatory and refractory diseases associated with leukocytes

Abstract:  Apheresis has been recognized both economically and therapeuticallyas a novel approach for the treatment of inflammatory diseases,and certain others, which respond poorly to drug therapy. This reportis about Adacolumn, an adsorptive carrier based granulocyteand monocyte apheresis device with a volume of 335 mL,filled with about 220 g of cellulose acetate beads of 2 mmdiameter as the column adsorptive carriers. Pre‐ and post‐columnleukocyte counts have shown that the carriers adsorb about 65% ofgranulocytes, 55% of monocytes and 2% of lymphocytesfrom the blood in the column. Additionally, after apheresis, thereis a marked decrease in inflammatory cytokines (TNF‐α,IL‐1β, IL‐6 and IL‐8) produced by blood leukocytes,together with down‐modulation of l‐selectinand the chemokine receptor CXCR3. Adacolumn has been used to treatpatients with rheumatoid arthritis, ulcerative colitis and HIV infection. Typicalapheresis sessions have been 4–10, at a frequency of oneor two sessions per week. Treatment of patients with Adacolumn hasbeen associated with very promising efficacy and safety data. Accordingly,in Japan, Adacolumn has been approved by the Ministry of Healthfor the treatment of ulcerative colitis. Furthermore, Adacolumnmet the required quality and safety standards for medical devices andreceived an EC certification (CE‐mark) from TUV in 1999. However,although Adacolumn carriers are very efficient in depleting excessand activated granulocytes and monocytes/macrophages, theclinical efficacy associated with Adacolumn apheresis cannot befully explained on the basis of reducing granulocytes and monocytesper se. Hence, a long lasting effect on inflammatory cytokine generation,chemokine activities or immunomodulation is likely, but the precisemechanisms involved are not fully understood yet.

Effects of pemirolast and tranilast on intimal thickening after arterial injury in the rat

We previously reported that tranilast, an antiallergic agent, reduced intimal thickening after endothelial injury in rats. In this study, to verify whether or not antiallergic agents inhibit intimal thickening, we investigated the effect of pemirolast on intimal thickening after endothelial injury and compared its effect with that of tranilast. Administration of two antiallergic agents, pemirolast (0.1, 1, and 10 mg/kg, p.o.) and tranilast (300 mg/kg, p.o., daily), was begun 2 days before endothelial injury and continued until the animals were killed. Endothelial injury in the rat femoral artery was induced by a photochemical reaction between localized irradiation by green light and intravenously administered rose bengal. To evaluate intimal hyperplasia, we measured the cross-sectional area of the intima 21 days after endothelial damage. Pemirolast at doses of 0.1, 1, and 10 mg/kg reduced the intimal area to 2.10 +/- 0.33, 1.36 +/- 0.19, and 1.35 +/- 0.18 (x0.01 mm2), respectively, and tranilast showed a tendency to reduce the intimal area, which was 1.86 +/- 0.35 x 0.01 mm2, compared with findings for controls (2.83 +/- 0.49 x 0.01 mm2). In rat A10 vascular smooth-muscle cells, we investigated the effects of antiallergic agents on migration by using a modified Boyden chamber assay and on proliferation by using the bromodeoxyuridine-incorporation assay. Two antiallergic agents inhibited in a concentration-dependent manner both migration and proliferation of smooth muscle cells stimulated by platelet-derived growth factor. These results suggest that antiallergic agents directly inhibit migration of smooth-muscle cells to the intima from the media and proliferation in the intima, and that pemirolast has more potent antihyperplastic action than does tranilast. Antiallergic agents may be effective in preventing restenosis after coronary angioplasty.

Essential in Vivo Roles of the C-type Lectin Receptor CLEC-2 EMBRYONIC/NEONATAL LETHALITY OF CLEC-2-DEFICIENT MICE BY BLOOD/LYMPHATIC MISCONNECTIONS AND IMPAIRED THROMBUS FORMATION OF CLEC-2-DEFICIENT PLATELETS

CLEC-2 has been described recently as playing crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis. The snake venom rhodocytin is known as a strong platelet activator, and we have shown that this effect is mediated by CLEC-2 (Suzuki-Inoue, K., Fuller, G. L., García, A., Eble, J. A., Pöhlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542–549). Podoplanin, which is expressed on the surface of tumor cells, is an endogenous ligand for CLEC-2 and facilitates tumor metastasis by inducing platelet aggregation. Mice deficient in podoplanin, which is also expressed on the surface of lymphatic endothelial cells, show abnormal patterns of lymphatic vessel formation. In this study, we report on the generation and phenotype of CLEC-2-deficient mice. These mice are lethal at the embryonic/neonatal stages associated with disorganized and blood-filled lymphatic vessels and severe edema. Moreover, by transplantation of fetal liver cells from Clec-2−/− or Clec-2+/+ embryos, we were able to demonstrate that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, possibly through homophilic interactions without apparent increase in bleeding tendency. We propose that CLEC-2 could be an ideal novel target protein for an anti-platelet drug, which inhibits pathological thrombus formation but not physiological hemostasis.

Pharmacokinetics of Voriconazole and Cytochrome p450 2C19 Genetic Status

gested and that possibly could be suitable for drugs eliminated by biliary excretion. Even though a specific classification is used for including subjects in the study, evaluation of data and dose recommendations may very well be based on relevant individual laboratory parameters (albumin levels, prothrombin time, bilirubin levels, alkaline phosphatase levels, fibrinogen levels, and so on). Sponsors and regulatory authorities also need to consider that the characteristics of the classification systems are not well known by the prescribers. In conclusion, we have presented several arguments disqualifying the Maddrey function as a general marker for hepatic function in pharmacokinetic trials. An inappropriate and routinelike classification of hepatic impairment may result in the selection of a study population that does not reflect the effects of reduced elimination capacity and a study with a low predictive value for patients at risk. Clearly, there is a need for further development with respect to what hepatic marker or classification systems to use in pharmacokinetic hepatic impairment studies. The pharmaceutical industry is encouraged to implement its knowledge of the mechanism of drug elimination into the design of the studies and to further explore hepatic markers that may be used for characterizing the pharmacokinetic profile of drugs dependent on hepatic function for their elimination.

The common gene variants of CYP2C19 affect pharmacokinetics and pharmacodynamics in an active metabolite of clopidogrel in healthy subjects.

EG, Mumford AD. Pharmacodynamic resistance to warfarin associated with a Val66Met substitution in vitamin K epoxide reductase complex subunit 1. Thromb Haemost 2005; 93: 23–6. 17 Wilms EB, Touw DJ, Conemans JM, Veldkamp R, Hermans M. A new VKORC1 allelic variant (p.Trp59Arg) in a patient with partial resistance to acenocoumarol and phenprocoumon. J Thromb Haemost 2008; 6: 1224–6. 18 Tie JK, Nicchitta C, von Heijne G, Stafford DW.Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/ cotranslocation. J Biol Chem 2005; 280: 16410–6. 19 Goodstadt L, Ponting CP. Vitamin K epoxide reductase : homology, active site and catalytic mechanism. Trends Biochem Sci 2004; 29: 289–92.

Inhibitory Effect of MCI-186, a Free Radical Scavenger, on Cerebral Ischemia Following Rat Middle Cerebral Artery Occlusion

1. In this study, we investigated the effect of a radical scavenger, MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one), on cerebral damage induced by rat middle cerebral artery (MCA) occlusion, and further measured the hydroxyl radical level at the ischemic border zone using a microdialysis technique. 2. MCI-186, at a dose of 3 mg/kg per 30 min, was administered as a continuous infusion two times for 30 min, starting 20 min and then 80 min after Rose Bengal injection. 3. MCI-186 significantly (P < 0.05) reduced size of cerebral damage 24 hr after MCA occlusion and significantly (P < 0.05) reduced hydroxyl radical level.

The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1.

In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1′: Arg346-Met347). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02–1.0 nm; half-maximal effect at ∼0.1 nm). Subtilisin NAT dose dependently (0.06–1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 ± 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 ± 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.

Evaluation of the combination of a tissue-type plasminogen activator, SUN9216, and a thromboxane A2 receptor antagonist, vapiprost, in a rat middle cerebral artery thrombosis model.

Background and Purpose We aimed to evaluate a modified tissue-type plasminogen activator, SUN9216, and the combination of SUN9216 and a thromboxane A2 receptor antagonist, vapiprost, in a rat middle cerebral artery thrombosis model. Methods Under anesthesia, the left middle cerebral artery was observed under an operation microscope without cutting the dura mater via a subtemporal craniotomy. Photoillumination (wave length, 540 nm) was applied to the middle cerebral artery, and then rose bengal (20 mg/kg) was administered intravenously. The reopening of the middle cerebral artery by SUN9216, injected 30 minutes after middle cerebral artery occlusion, was observed under an operation microscope for a 60-minute observation period. Twenty-four hours after the operation, sections of the cerebrum were stained with triphenyltetrazolium chloride, and the area of cerebral infarction was analyzed by a computer. Results The combination of SUN9216 and vapiprost caused reopening of the middle cerebral artery in 58.8% of the rats, which was a greater percentage than that achieved with SUN9216 alone (31.6%). In contrast, saline did not cause reopening of the middle cerebral artery during the 60-minute observation period. The area of cerebral infarction in rats reperfused with SUN9216 was significantly reduced compared with that in the control group. The infarction area in rats treated with the combination of SUN9216 and vapiprost was reduced compared with that in rats treated with SUN9216 alone; this was the case whether or not the occlusion was reperfused. There was a significant correlation between the time of reopening of the middle cerebral artery and area of cerebral infarction. Conclusions A single injection of SUN9216 was effective in recanalizing the vessel and reducing the area of cerebral infarction.

PAI-1, progress in understanding the clinical problem and its aetiology

Plasminogen activator inhibitor‐1 (PAI‐1, also known as SERPINE1) is a member of the serine protease inhibitor (SERPIN) superfamily and is the primary physiological regulator of urokinase‐type plasminogen activator (uPA) and tissue‐type plasminogen activator (tPA) activity. Although the principal function of PAI‐1 is the inhibition of fibrinolysis, PAI‐1 possesses pleiotropic functions besides haemostasis. In the quarter century since its discovery, a number of studies have focused on improving our understanding of PAI‐1 functions in vivo and in vitro. The use of Serpine1‐deficient mice has particularly enhanced our understanding of the functions of PAI‐1 in various physiological and pathophysiological conditions. In this review, the results of recent studies on PAI‐1 and its role in clinical conditions are discussed.

Stromelysin-1 (MMP-3) is critical for intracranial bleeding after t-PA treatment of stroke in mice

Summary.  Background: Tissue‐type plasminogen activator (t‐PA) is approved for treatment of ischemic stroke patients, but it may increase the risk of intracranial bleeding (ICB). Matrix metalloproteinases (MMPs), which can be activated through the plasminogen/plasmin system, may contribute to ICB after ischemic stroke. Objectives: To explore the contribution of plasminogen, MMP‐3 and MMP‐9 to ICB associated with t‐PA treatment after ischemic stroke. Methods: Using a thrombotic middle cerebral artery occlusion (MCA‐O) model, ICB was studied in mice with genetic deficiencies of plasminogen (Plg−/−), stromelysin‐1 (MMP‐3−/−), or gelatinase B (MMP‐9−/−) and their corresponding wild‐type (WT) littermates. The induction of MMP‐3 and MMP‐9 was also studied in C57BL/6 WT mice. Results: ICB induced by t‐PA (10 mg kg−1) was significantly less than WT in Plg−/− (P < 0.05) and MMP‐3−/− (P < 0.05) but not in MMP‐9−/− mice. Furthermore, administration of the broad‐spectrum MMP inhibitor GM6001 after t‐PA treatment reduced ICB significantly (P < 0.05) in MMP‐3+/+ mice, but had no effect on MMP‐3−/− mice. MMP‐3 expression was significantly enhanced at the ischemic hemisphere; with placebo treatment, it was expressed only in neurons, whereas it was up‐regulated in endothelial cells with t‐PA treatment. Although MMP‐9 expression was also significantly enhanced at the ischemic brain, the amount and the distribution were comparable in mice with and without t‐PA treatment. Conclusions: Our data with gene‐deficient mice thus suggest that plasminogen and MMP‐3 are relatively more important than MMP‐9 for the increased ICB induced by t‐PA treatment of ischemic stroke.