Toshiya Minagawa

Effect of a New Inhibitor of the Synthesis of 20-HETE on Cerebral Ischemia Reperfusion Injury

Background and Purpose— Arachidonic acid that is released following cerebral ischemia can be metabolized to 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a potent vasoconstrictor that may contribute to ischemic injury. This study examined the effects of blockading the synthesis of 20-HETE with TS-011 on infarct size after transient occlusion of the middle cerebral artery (MCAO) of rats and after thromboembolic stroke in monkeys. Methods— Rats were treated with TS-011 or vehicle at various times after MCAO. Infarct size was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining and plasma levels of 20-HETE were determined by liquid chromatography mass spectrometry (LC/MS). The effect of TS-011 on infarct size was also studied in monkeys after introduction of a clot into the internal carotid artery. Results— Plasma levels of 20-HETE increased after MCAO in rats. TS-011 (0.01 to 1.0 mg/kg per hour) reduced infarct volume by 40%. Chronic administration of TS-011 for 7 days reduced neurological deficits after MCAO in rats. TS-011 given in combination with tissue plasminogen activator also improved neurological outcome in the stroke model in monkeys. Conclusion— These results suggest that blockade of the formation of 20-HETE with TS-011 may be useful for the treatment of ischemic stroke.

Continuous inhibition of 20-HETE synthesis by TS-011 improves neurological and functional outcomes after transient focal cerebral ischemia in rats

TS-011, a potent and selective inhibitor of 20-HETE synthesis, has been described as providing significant benefits in animal stroke models. However, no studies have investigated changes in brain 20-HETE levels after cerebral ischemia. Also lacking are studies of TS-011 pharmacodynamics with respect to brain 20-HETE levels that may explain the benefits of TS-011 in animal models of ischemic stroke. The present study sought to explore changes in 20-HETE levels in brain tissue, as well as in plasma, after a 90-min episode of transient focal cerebral ischemia. Pharmacodynamics of TS-011 were also examined. Then, we evaluated the long-term effects of TS-011 when administered as in this pharmacodynamics study. The major findings of the present study are as follows: (1) brain 20-HETE levels increased significantly within 7.5h after MCAO; (2) TS-011 at doses of 0.1 and 0.3mg/kg administered at regular 6-h intervals appeared to reduce brain 20-HETE levels continuously; (3) TS-011 when administered as in this pharmacodynamics study improved long-term neurological and functional outcomes. These findings strongly suggest that 20-HETE plays an important role in the development of neurological and functional deficits after focal cerebral ischemia and that TS-011 may provide benefits in patients suffering ischemic stroke.

Blood-brain-barrier Transport of Lipid Microspheres Containing Clinprost, a Prostaglandin I2 Analogue

Because the permeability of the blood‐brain barrier to lipid microspheres (LMs) has not hitherto been demonstrated, blood‐brain‐barrier permeability to LM containing the prostaglandin I2 analogue clinprost has been evaluated for an in‐vitro system of primary cultured monolayers of bovine brain capillary endothelial cells (BCECs), by a capillary depletion study in rats and by an in‐situ brain perfusion study in normal and 4‐vessel‐occluded fore brain ischaemic rats.

Entrapping Efficiency and Drug Release Profile of an Oil-in-Water (o/w) Emulsion Formulation Using a Polydimethylsiloxane-Coated Glass Bead Assay

Evaluation of entrapping efficiency is difficult for an o/w emulsion formulation containing a lipophilic oily drug, isocarbacyclin methyl ester (TEI-9090), by commonly employed techniques (dialysis, ultrafiltration, or gel filtration), because of its adsorption to the system materials. Employing this characteristic of TEI-9090, we developed an adsorption technique with polydimethylsiloxane-coated glass beads (PDMS-GB). The assay is based on the quantitative adsorption of unentrapped TEI-9090 to the PDMS-GB. The entrapping efficiency of a 10% soybean oil emulsion containing [3H]TEI-9090 (1 µg/mL) assayed by this method approached 100%. The PDMS-GB assay was performed for the emulsion diluted 100 times with physiological saline at different time intervals after dilution over a period of 24 hr. A plot of [3H]TEI-9090 in the emulsion particles versus time showed rapid release within 1 hr, followed by very slow release, reaching equilibrium. Applying first-order kinetics, the data were found to fit to a biexponential equation over the first hour of release. The terminal release resembled the first-order release of the drug from the phospholipid-rich infranatant, which was separated from the creamy layer by ultracentrifugation of the emulsion and contained 35% [3H]TEI-9090. These results suggest that the drug is released from two components in the emulsion.

Determination of protein binding of a highly lipophilic drug, isocarbacyclin methyl ester (TEI-9090), using a polydimethylsiloxane-coated glass beads assay.

Abstract— An adsorption technique with polydimethylsiloxane‐coated glass beads (PDMS‐GB) was developed to determine the protein binding of a highly lipophilic and hydrophobic drug. The present assay method is based on the quantitative adsorption of unbound drug to the PDMS‐GB. This method of batch separation in a glass assay tube has an advantage of simplicity and rapidity. To evaluate the reliability of PDMS‐GB assay, we compared the protein binding of diazepam in serum in‐vitro measured by ultrafiltration and PDMS‐GB assay. There was no significant difference between the extent of binding measured by each method. Using PDMS‐GB assay, we determined the protein binding of the prostaglandin I2 (PGI2) analogue isocarbacyclin methyl ester (TEI‐9090), whose binding cannot be measured by commonly employed techniques (equilibrium dialysis, ultrafiltration, gel filtration or ultracentrifugation) because of a high degree of adsorption to membranes, resins or tubes. The percentage of TEI‐9090 bound in human serum, 4% human serum albumin (HSA, fatty acid‐free) and dog serum were ∼98, ∼87 and ∼95%, respectively, and these values were independent of TEI‐9090 concentration up to 10 ng mL−1. The binding of isocarbacyclin (TEI‐7165) to serum protein in man, dogs, rabbits and rats, determined by ultrafiltration, was also high (>90%). While the displacement of TEI‐9090 and TEI‐7165 binding to HSA by aspirin, salicylic acid and indomethacin was not observed, clofibric acid and free fatty acids significantly inhibited the protein binding of both compounds. These results indicate that the binding site of TEI‐9090 and TEI‐7165 on HSA could be identical with the possible binding site of PGI2.

Identification of 2-[2-(4-tert-butylphenyl)ethyl]-N-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (29) as an orally available MGAT2 inhibitor.

MGAT2 (monoacylglycerol acyltransferase 2) is expected to be an attractive target for the drug treatment of obesity, diabetes, and other disease. We describe our exploration and structure-activity relationship (SAR) study of 2,3-dihydro-1H-isoindole-5-sulfonamide derivatives. In this study, we identified 29 as an orally available inhibitor of MGAT2 through optimization especially in terms of solubility. This compound exhibited moderate potency in the enzyme inhibitory assay (IC50 = 1522 nM) and significant suppression of fat absorption (57% inhibition) in mice oral lipid tolerance test.

Metabolic fate of midodrine. (II). Distribution and protein binding.

Tissue distribution of 14C-midodrine and its active metabolite 14C-DMAE was investigated after oral and intravenous administration to rats using autoradiography and scintillation counting. Serum protein binding was also examined. After oral administration of 14C-midodrine at a dose of 3.4 μmol/kg, the radioactivity was distributed extensively throughout the body except for the central nervous system, testis and fat. High concentration of radioactivity was found in the gastrointestinal tract, liver and kidney followed by the lung, spleen, hypophysis and adrenal. The levels in these tissues were higher than that in the plasma. Elimination of radioactivity from tissues paralleled that of plasma, and no marked accumulation in any tissue was found. Although the tissue concentrations in early time after oral administration of 14C-midodrine were higher than that in the case of 14C-DMAE, the distribution profiles were essentially similar in each case. However, 5 min after intravenous administration of 14C-midodrine or 14C-DMAE, the distribution profiles were found to be different from each other. DMAE and other metabolites were found in the tissues after the administration of 14C-midodrine and only a small amount of unchanged drug was detected. Serum protein binding of 14C-midodrine or 14C-DMAE in vitro was 10 ?? 20 % in rats, rabbits and dogs, and 24 ?? 31 % in human. The extent of serum protein binding of radioactive substances was about 22 % at 0.5 hr after oral administration of 14C-midodrine to rats and was increased with time.