Cilostazol stimulates revascularisation in response to ischaemia via an eNOS-dependent mechanism.

OBJECTIVES: Cilostazol is known to be a selective inhibitor of phosphodiesterase 3 and is generally used to treat intermittent claudication caused by peripheral arterial disease. However, there is little information concerning the effect of cilostazol on angiogenesis. Here, we investigated whether cilostazol modulates the angiogenic process in vivo employing a hindlimb model of ischaemia-induced angiogenesis. DESIGN: This was an experimental study. MATERIALS AND METHODS: Wild-type (WT) mice were randomly divided into two groups and were treated with or without cilostazol. One week later, the mice were subjected to unilateral hindlimb ischaemia. Angiogenesis was determined by laser Doppler analysis and capillary density stained with CD31. The expression of endothelial nitric oxide synthase (eNOS) was assessed by immunoblotting. RESULTS: WT mice treated with cilostazol showed accelerated neo-vascularisation following hindlimb ischaemic surgery on post-operative day 14 based upon laser Doppler measurements of blood flow (cilostazol-treated group, 0.54 ± 0.13 vs. control group, 0.38 ± 0.11; P-<-0.05). The capillary density in the ischaemic hindlimb was also significantly greater in WT mice treated with cilostazol than in non-treated WT mice (cilostazol-treated group, 1.63 ± 0.10 vs. control group, 1.15 ± 0.12; P-<-0.01). Cilostazol stimulated an ischaemia-induced increase in the phosphorylation of eNOS in the ischaemic limbs. Administration of NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) abolished cilostazol-induced increase in limb perfusion. CONCLUSIONS: Our observations indicate that cilostazol can promote neo-vascularisation in response to tissue ischaemia via an eNOS-dependent mechanism. Cilostazol could be useful for treatment of ischaemic limb diseases.

Separate evaluation of intestinal and hepatic metabolism of three benzodiazepines in rats with cannulated portal and jugular veins: comparison with the profile in non-cannulated mice.

Pharmacokinetic analyses of three kinds of benzodiazepines--midazolam (MDZ), triazolam (TRZ) and alprezolam (APZ)--were performed in rats with cannulated portal and jugular veins. Each drug was administered to the double-cannulated rats, and pharmacokinetic data for the parent drugs and their 1'- and 4-hydroxylated metabolites were compared with those obtained in non-cannulated mice. In bioavailability, the drugs ranked APZ >> TRZ = MDZ in rats, and APZ > TRZ >> MDZ in mice, with the values for MDZ remarkably different between rats and mice (19% in rats versus 2.3% in mice). In contrast, hepatic availability (Fh) was similar (APZ > TRZ > MDZ) in both species. Highly significant relationships were found between the ratio of the area under the plasma concentration-time curve (AUC) for the parent drugs in portal blood (AUC(por)) to that in systemic blood (AUC(sys)) and Fh in rats and mice. The double-cannulated rat is useful for estimating the hepatic availability of drug candidates by determining the AUC values for the parent drugs in portal and systemic blood samples.

Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity.

Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 microM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.

Cerebral oxygen metabolism monitoring under hypothermia for severe subarachnoid hemorrhage: report of eight cases.

OBJECTIVES: The significance of cerebral oxygen metabolism monitoring under hypothermia for severe subarachnoid hemorrhage (SAH) was studied. MATERIAL AND METHODS: Cerebral oxygen metabolism monitoring (jugular venous oxygen saturation: SjO2, arterio-jugular venous difference of oxygen: AJDO2, and oxygen extraction fraction:OEF) during hypothermia (32-34 degrees C) was evaluated in eight patients with SAH (severe vasoapasm group: five patients, and severe brain damage group: three patients). RESULTS: In favorable cases in both groups, each parameter tended to normalize during hypothermia therapy. When changes in SjO2 were normal, however, the value of AJDO2 was low in unfavorable cases in the severe vasospasm group. In unfavorable cases in the severe brain damage group, high level of SjO2 and low level of OEF and AJDO2 were shown even if hypothermia therapy was performed. CONCLUSIONS: The measurement of SjO2 and AJDO2 is useful for estimation of cerebral oxygen metabolism in patients with severe conscious disturbance after SAH under hypothermia therapy.

[Chemotherapy for gliomas based on the expression levels of drug resistant genes].

Drug resistance, which often occurs during chemotherapy, is still a great obstacle to the success of human malignancy treatment. Among many possible mechanisms of drug resistance (biological, biochemical, kinetic or pharmacological), both typical and atypical multidrug-resistance (MDR) have been extensively studied. We picked up MDR-1, MXR, MRP1, MRP2, TopoII alpha, MGMT, and GST-pi as drug-resistant gene, based on experimental data and previous reports. Expression of these genes were measured in 14 malignant glioma specimens by reverse transcription polymerase chain reaction assay. We chose anticancer drugs for each patient, based on results of drug resistant gene expression to acquire good response to drugs. Though our follow-up periods are not long enough to analyze the results of our chemotherapy, 78% (7/9) of our glioma patients who were treated with our chemotherapy are free from tumor progression. The assays, which measure the expression of drug resistant genes, are necessary to allow rapid detection of the drug-sensitivity to chemotherapy in malignant glioma patients.

Repeated thalamic haemorrhage: case report and review of the literature.

Recurrence of thalamic haemorrhage has rarely been reported. A 70-year-old woman had recurrent thalamic haemorrhage five-times during a period of 6 years. The first, second and fifth haemorrhages were located in the right thalamic region, and the third and fourth haemorrhages in the left thalamic region. Cranial computed tomography and magnetic resonance imaging revealed no abnormal lesion. After the first, second, and third haemorrhage with medical treatments, the patient recovered her functional ability or was at least capable of self-care at home. However, after the fourth and fifth haemorrhage, with medical therapy the patient's prognosis was severe disability. In this case, systemic blood pressure was normalized without antihypertensive agents after the first attack. However, there was an episode of sudden hypertension at each attack. Although the mechanism of rebleeding has not been clarified, rebleeding might be associated with changes of cerebral circulation following the previous haemorrhage.

Screening for phenoloxidases from edible mushrooms.

A screening test for phenoloxidases from edible mushrooms was done on potato dextrose agar plates that contained phenolic chemicals. Many edible mushrooms showed positive reactions on the agar plates. Among them, Auricularia auricula-judae, Clitocybe nebularis, Lentinus edodes, Pholiota aurivella, and Pseudohiatula oshimae produced a considerable amount of phenoloxidases, and these enzymes showed maximum activities in the acidic pH region.

Expression of DNA topoisomerase I, IIalpha, and IIbeta in human brain tumors.

We investigated the expression of DNA topoisomerase I (Topo I), IIalpha (Topo IIalpha), and IIbeta (Topo IIbeta) mRNA using reverse transcription-polymerase chain reaction (RT-PCR) assay in 31 human brain tumors, and examined the relationship between DNA topoisomerase mRNA expression and Topo IIalpha and MIB-1 positive index (PI) as a cell proliferation marker. Topo IIalpha mRNA was expressed in 11 of 31 cases, and Topo I and IIbeta were each expressed in 18 of 31 cases. A significant correlation was seen between the MIB-1 PI and Topo IIalpha PI (P < 0.001). The cases with overexpression of Topo IIalpha mRNA had significantly high MIB-1 and Topo IIalpha PI (P < 0.0001). There was no significant correlation between Topo I and IIbeta mRNA expression and MIB-1 PI. We concluded that it was useful as a cell proliferation marker to analyze the expression of Topo IIalpha mRNA using RT-PCR in human brain tumors.

Effects of delayed intraischemic and postischemic hypothermia on a focal model of transient cerebral ischemia in rats.

BACKGROUND AND PURPOSE: Intraischemic mild hypothermia has been shown to be neuroprotective in reducing cerebral infarction in transient focal ischemia. As a more clinical relevant issue, we investigated the effect of delayed intraischemic and postischemic hypothermia on cerebral infarction in a rat model of reversible focal ischemia. We also examined the effect of hypothermia on the inflammatory response after ischemia-reperfusion to assess the neuroprotective mechanism of brain hypothermia. METHODS: Rats were subjected to 2 hours of middle cerebral artery occlusion followed by 22 hours of reperfusion under the following protocols: (1) rats were treated with normothermia (37.0 degrees C, 4 hours) and then housed in room temperature (25 degrees C, 18 hours) and (2) rats were treated with hypothermia (33.0 degrees C, 4 hours, brain temperature modulation was started 30 minutes before the reperfusion) and then housed in cold temperature (5 degrees C, 18 hours). Animals were killed 24 hours after the onset of ischemia. The infarct volume was examined with 2,3,5-triphenyl-tetrazolium chloride staining. The accumulation of polymorphonuclear leukocytes (PMNLs) and the expression of intercellular adhesion molecule-1 mRNA were evaluated in both groups. RESULTS: A significant reduction (P<0.05) in infarct volume was found in the hypothermia group compared with the normothermia group. Compared with the normothermia group, hypothermic treatment also significantly reduced the accumulation of PMNLs (P<0.01) and inhibited the overexpression of intercellular adhesion molecule-1 mRNA at 22 hours of reperfusion after 2 hours of ischemia. CONCLUSIONS: Ischemic brain damage can be reduced with delayed intraischemic and prolonged postischemic hypothermia in a focal model of transient cerebral ischemia in rats. The neuroprotective mechanism of hypothermia may be mediated by suppression of PMNL-mediated inflammatory response after ischemia-reperfusion in this model.

DNA topoisomerase IIalpha protein and mRNA expression in intracranial meningiomas.

We examined the expression of DNA topoisomerase IIalpha (Topo IIalpha) immunohistochemically using a monoclonal antibody and compared its proliferative potential [MIB-1 labeling index (LI)] and recurrence to verify the possible influence of Topo IIalpha on the progress of meningiomas. The reverse transcription-polymerase chain reaction (RT-PCR) assay was also performed to evaluate the expression of Topo IIalpha mRNA. Formalin-fixed, paraffin-embedded tissue sections of 52 meningiomas (18 meningothelial types, 16 fibrous types, 4 transitional types, 4 psammomatous types, 1 angiomatous type, 1 secretory type, 5 atypical types, and 3 anaplastic types) were used for immunostaining. The Topo IIalpha labeling index (LI) was 1.4 +/- 1.9% (mean +/- SE) in benign meningiomas and 4.5 +/- 1.6% in atypical or anaplastic meningiomas, representing significant differences between them (P < 0.0001). RT-PCR assay revealed that Topo IIalpha mRNA expression was associated with Topo IIalpha LI. A significant correlation was seen between Topo IIalpha LI and MIB-1 LI (r = 0.517; P < 0.01). Recurrence was significantly more frequent in patients with more than 1.5% of Topo IIalpha LI than in those with 1.5% or less (P < 0.005). In conclusion, Topo IIalpha protein and mRNA expression correlated with clinical malignancy and the potential for predicting the regrowth of meningiomas.

[Incidence of mutation and deletion in topoisomerase II mRNA of etoposide and m-AMSA resistant cell lines].

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. To further understand resistance to topoisomerase (topo) II inhibitors, 50 sublines were isolated as single clones from parental cells by exposure to ETP or m-AMSA. Subsequently, a population of cells from each subline was exposed to three-fold higher drug concentrations allowing 16 stable sublines to be established at higher extracellular drug concentration. The frequency and nature of mutations in topo II in the drug selected cell lines have been evaluated. In order to screen a large number of cell lines, an RNase protection assay was developed. Fragments covering the entire coding sequence of topo II was isolated after PCR amplification and subcloned in pGEM3Z vector. Using this approach, mismatches was observed in 13.6% of resistant cell lines (12% of resistant cell lines exposed to lower drug concentrations and 18.8% of resistant cell lines exposed to higher drug concentrations). Our findings suggest that mutations of topo II gene seem to be an important and frequent mechanism of resistance to topo II inhibitors.

Design of novel hybrid vitamin C derivatives: thermal stability and biological activity.

Novel hybrid L-ascorbic acid (vitamin C) derivatives with other biologically active substances, 5-hydroxy-2-hydroxymethyl-beta-pyrone (kojic acid) and alpha-tocopherol (vitamin E), linked at the C-2 or C-3 hydroxyl group were synthesized, and their thermal stability and inhibitory effects on tyrosinase activity, active oxygen species (AOS), and free radicals were estimated in vitro. It was found that a hydrophilic derivative, 2-O-(5-hydroxy-4H-pyran-4-one-2-methyl)-L-ascorbic acid (1), exhibited good thermal stability and inhibitory activities against tyrosinase catalyzed melanin formation, AOS, and free radicals compared to vitamin C and its conventional derivatives (such as the 2-phosphate 6-stearate and 2.6-dipalmitate, and 2-O-octadecylascorbic acid), as well as vitamin E, kojic acid, and arbutin. It is apparent that 1 has the biological properties of vitamin C and kojic acid, and acts synergistically. The hydroxyl groups at the C-3 position of the vitamin C moiety and the C-5 position of the kojic acid moiety are critical for the biological activities. We consider that the kojic acid moiety of 1 counterbalances the diminution of the biological activity due to shielding of the biologically important C-2 hydroxyl group of the vitamin C moiety. In addition, the thermal stability was significantly improved relative to not only vitamin C but also kojic acid. Further, a lipophilic derivative, 3-O-[(alpha-tocopheryloxy)-2-hydroxypropyl]-L-ascorbic acid, 2, was far more stable than vitamin C and its typical lipophilic derivatives. Compound 2 exhibited almost the same inhibitory activities against tyrosinase-catalyzed melanin formation, AOS, and free radicals as typical lipophilic derivatives, although these biological activities of 2 were lower than those of vitamin C.

Synthesis of novel vitamin C phosphodiesters: stability and antioxidant activity.

A novel series of hybrid L-ascorbic acid (vitamin C) phosphodiesters linked at the C-2 hydroxyl group with other biologically active substances, namely myo-inositol, arbutin, 4-hydroxy-L-proline, and glycolic acid were synthesized, and their thermal stability and reducing activity against free radicals were estimated in vitro. All of the phosphodiesters exhibited high thermal stabilities; however, their antioxidant activities in vitro were generally lower than that of vitamin C.

A putative lipophilic stimulant carrier protein commonly found in the taste and olfactory systems. A unique member of the pheromone-binding protein superfamily.

In chemosensory systems, a variety of lipophilic ligand-binding proteins have been found in saliva or nasal mucus. Lipophilic stimulants reach the receptor membrane, carried by these proteins. An acidic 14-kDa protein purified in the blowfly, Phormia regina, belongs to the insect pheromone-binding protein superfamily, but unlike other lipophilic ligand-binding proteins in insect or vertebrate chemosensory systems, it was distributed in both taste and olfactory organs. A similar protein was also isolated in Drosophila melanogaster. Considering their distributions, cDNA sequences and structural features, we concluded that these proteins belong to a unique subfamily whose members have convergently evolved for a common function required for both senses of taste and olfaction. By an electrophysiological experiment using antiserum, we also suggested that these proteins carry fragrant components of natural foods in taste systems as well as in olfactory systems.

Local drug delivery systems for the treatment of periodontal disease.

Filmy local drug delivery systems (LDDSs) were administered to periodontal pockets in beagles with induced periodontitis, and the in vivo-in vitro correlation of drug release from the LDDS and changes in the clindamycin (CLDM) concentration in the periodontal pocket fluid were studied. The in vitro drug release rate from the LDDS was determined by the dissolution study, without agitation, using phosphate buffer as the dissolution medium at 37 degrees C, and the in vivo drug release rate was determined according to the decrease in the drug load remaining in the LDDS after administration in periodontal pockets. The in vivo drug release rate from LDDSs was lower than the in vitro rate determined by the dissolution study, but the two rates showed a correlation in LDDSs that released drugs by diffusion. Therefore, the in vivo drug release rate was considered to be estimated from the results of the in vitro dissolution study. Changes in the drug concentration in the periodontal pocket fluid after administration of LDDS were dependent on the drug release properties of the LDDS. Also, when CLDM was administered as an aqueous solution in periodontal pockets, its concentration in the periodontal pocket fluid decreased according to a pseudo first-order equation. Therefore, the concentration in the periodontal pocket fluid after administration of a LDDS is considered to be simulated by the one compartment model based on a pseudo first-order elimination process.

Local ofloxacin delivery using a controlled-release insert (PT-01) in the human periodontal pocket.

PT-01, a controlled-release insert, was developed for topical chemotherapy in periodontal disease. It is a soluble insert that consists of fast-release and sustained-release parts containing ofloxacin (OFLX) as an antibacterial agent. In this study, the release profile of OFLX from PT-01 was investigated in vitro. Twelve adult volunteers were administered OFLX as PT-01 or as an aqueous solution into their periodontal pockets, OFLX concentrations in gingival crevicular fluid (GCF) were evaluated from the viewpoint of pharmacokinetics. The in vitro release profile of OFLX from PT-01 showed a biphasic pattern. The release rate of OFLX was relatively rapid in the early phase and slow thereafter. When OFLX aqueous solution was administered into periodontal pockets, the OFLX level in GCF rapidly decreased to be about 1/100 after 30 minutes. When PT-01 was inserted into the pockets, the OFLX level in GCF immediately reached a peak (about 12 mg/ml), and gradually decreased until the 3rd day, and maintained a constant level above 2 micrograms/ml, the effective minimum antibacterial concentration for periodontopathic microorganisms, from the 3rd to 7th day after insertion. No side-effects were observed in the volunteers who received the PT-01 insert. The above results suggest that PT-01 is a suitable pharmaceutical preparation for periodontal chemotherapy.

Effects of Tween 80 and liposomes on the corneal permeability of anti-inflammatory steroids.

The effects of Tween 80 and liposomes on the corneal permeability of dexamethasone (DM) and dexamethasone valerate (DV) were investigated in vitro. A model based on diffusion theory could successfully be applied to analyzing the process by which DM penetrates the cornea from DM preparations. The penetration rate increased according to the concentration of free DM. Although the penetration rate was increased by pretreatment with Tween 80, it was not affected by pretreatment with liposomes. The steroid that penetrated through the cornea from DV preparations had been metabolized to DM. The transfer of DM across the cornea from DV preparations tended to slow down to some extent during the penetration process. Diffusion models were not applicable to this process, in contrast to the corneal penetration of DM from DM preparations. When liposome preparations of DV were applied, the penetration rate of DM across the cornea depended the concentration of free DV in these preparations. When DV aqueous preparations containing different concentrations of Tween 80 were applied, the penetration rate increased as the concentration of the surfactant was increased, even though the concentration of free DV in the suspensions was kept constant. These results suggest that the corneal permeability of anti-inflammatory steroids is not affected by liposomes, but is accelerated by Tween 80.