Ken-ichi Kohno

Ibudilast, an anti-allergic and cerebral vasodilator, modulates superoxide production in human neutrophils

We evaluated the effect of ibudilast on superoxide generation in human neutrophils by chemiluminescence development using luciferine analog, FCLA. By incubating neutrophils with ibudilast (2-200 microM) for more than 10 minutes, fMLP- or PMA-induced chemiluminescence was enhanced. However, the fMLP-induced chemiluminescence was suppressed by incubation for less than 10 minutes. This suppressed effect was missing with PMA-induced chemiluminescence. On the both fMLP- and PMA-induced chemiluminescence, the priming effect of ibudilast was further enhanced by the treatment with H-7, a protein kinase C inhibitor. In contrast, the priming effect of ibudilast on the fMLP-induced chemiluminescence was abolished by the treatment with ST-638, a selective inhibitor of tyrosine kinase. Ibudilast showed a transient stimulatory effect on cyclic AMP accumulation which continued for only a few minutes. Ibudilast showed no significant effect on phospholipase D dependent chemiluminescence, 1,4,5 trisphosphate level, or protein kinase C activity. Ibudilast inhibited extracellular calcium influx. These results suggest that ibudilast acts on or through tyrosine kinase to achieved its priming effect on the fMLP-induced chemiluminescence. The early and transient increase in cyclic AMP level may explain the inhibitory effect of ibudilast on the fMLP-induced chemiluminescence after short time of incubation.

A proposal for purification of salivary polymorphonuclear leukocytes by combination of nylon mesh filtration and density-gradient method: A validation by superoxide- and cyclic AMP-generating responses

Isolation and purification of salivary polymorphonuclear leukocytes (SPMNs) from accompanying epithelial cells was presented by using a density-gradient method with Ficoll. SPMNs samples prepared by already established methods (nylon mesh filtration) was compared with SPMNs samples after further purification by Ficoll (d = 1.083). Microscopically, SPMNs samples after nylon mesh filtration contain higher percentage of epithelial cells than SPMNs samples after Ficoll centrifugation. In response to stimulation of superoxide generation, both samples showed the same pattern of response. However, in response to forskolin and prostaglandin E1, cyclic AMP levels in samples after nylon mesh purification were significantly higher than in samples after Ficoll purification because of the presence of contaminating epithelial cells. We can conclude that, although nylon mesh filtration is satisfactory when we need to examine superoxide generation but further purification is necessary when we want to measure factors like intracellular cyclic AMP formation.

Comparison of susceptibility to apoptosis induced by rhTNF-α and cycloheximide between human circulating and exudated neutrophils

To determine whether exudated neutrophils differ from circulating ones in their apoptosis, rhTNF-alpha plus cycloheximide-induced apoptosis in human salivary neutrophils was compared to that in human neutrophils in peripheral blood. Concomitant treatment of peripheral blood neutrophils with rhTNF-alpha and cycloheximide-induced apoptosis in blood neutrophils within 3 hr, as evaluated both by light microscopic changes characteristic to apoptosis and by DNA fragmentation, whereas the same treatment failed to induce any apoptosis in salivary neutrophils. These results indicate that the exudation of neutrophils from blood into tissue is associated with marked changes in their functions such as alteration in their sensitivity to apoptosis-inducing stimuli.

Migration of neutrophils from blood to tissue: Alteration of modulatory effects of prostanoid on superoxide generation in rabbits and humans

Alteration of neutrophil function is associated with their migration from blood into tissue. We evaluated this alteration in both human and rabbit neutrophils, by comparing the inhibitory effects of prostanoids on formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide generation in human circulating blood neutrophils with those in saliva, and also comparing rabbit circulating blood neutrophils with those exudated into peritoneal cavity. We showed that EP-receptor agonists (PGE1) EP2/EP3 agonist (misoprostol), EP2-receptor agonist (butaprost) and DP-receptor agonist (PGD2) inhibited fMLP-stimulated superoxide production from human blood neutrophils in a concentration-dependent manner. In contrast, these prostanoids produced a significantly smaller maximum inhibition of fMLP-stimulated superoxide production in salivary neutrophils compared to those in blood neutrophils. Similar differences were observed for rabbit blood and peritoneal neutrophils. The inhibitory effect of EP2 agonist (butaprost) on the fMLP-stimulated superoxide generation in human blood neutrophils was significantly higher than that of EP3 agonist (ONO-AP-324). The EP1 antagonist (SC-51322) and EP4 antagonist (AH23848B) employed in this study could not antagonize the inhibitory effect of PGE2. TP agonist (U-46619) failed to show any inhibitory effect in either blood or salivary neutrophils. These results indicated that EP2 and DP receptors are the primary receptors mediating the prostanoids inhibition of fMLP-stimulated superoxide generation from neutrophils. Furthermore, it can be concluded that neutrophils become less responsive to prostanoids in terms of fMLP-stimulated superoxide production in association with their migration from blood to tissue.

Pharmacokinetic and Pharmacodynamic Properties of a New Thromboxane Receptor Antagonist (Z‐335) after Single and Multiple Oral Administrations to Healthy Volunteers

The pharmacokinetics and pharmacodynamics of a new oral thromboxane (TX) A2 receptor antagonist, Z‐335, were studied in healthy male volunteers following single doses (0.5–40 mg, PO) in a dose‐escalating manner and multiple doses (40 mg, PO, once daily for 7 consecutive days) with a single‐blind, placebo‐controlled design. Serial blood and urine samples were analyzed for Z‐335 and its metabolites to obtain key pharmacokinetic parameters. In the single‐dose (10, 20, and 40 mg) study, the maximum plasma concentration (Cmax) and area under the plasma concentration versus time curve (AUC) increased in proportion to the dose when administered after fasting, while the mean elimination half‐life (t1/2β) was essentially unchanged (7.79‐7.93 h). Recovery of the unchanged and taurine‐conjugated drugs in the urine within 24 hours was 6.5% to 8.4% and 11.9% to 14.2%, respectively. These parameters essentially remained unchanged when the effect of meal intake was evaluated at the dose of 20 mg with a crossover design. Ex vivo platelet aggregation in the plasma by a TXA2 analogue, U46619, was completely inhibited within 2 hours after all doses, and complete inhibition was maintained for 12 to 14 hours, depending on the dose. The aggregation induced by collagen was also inhibited to a lesser extent, whereas that by adenosine diphosphate was hardly influenced. In themultiple‐dose study, Cmax and AUC0–24 were increased by 34% after the last dose compared with the first dose. Z‐335 afforded extensive inhibition of platelet aggregation by U46619 throughout the administration period, which returned, however, almost to the control level 48 hours after the last dose. The agent was well tolerated without any abnormalities in subjective and objective symptoms, blood biochemistry, hematology, and urinalysis definitely attributable to the agent, except for the changes expected from its TXA2 receptor‐antagonizing actions. Z‐335 was concluded to be safe and to provide long‐lasting blockade of TXA2 receptors on the basis of a once‐daily regimen, promoting further clinical evaluation.

Role of nitric oxide in the convulsive seizures induced by fluoroquinolones coadministered with 4-biphenyl acetic acid

1. Contribution of nitric oxide to the convulsive seizures induced by fluoroquinolones (FQs) coadministered with 4-biphenyl acetic acid (BPAA), the active metabolite of fenbufen, was assessed in mice. 2. Enoxacin + 4-biphenyl acetic acid caused clonic seizures in all treated mice, followed by tonic seizures and death. These events were associated with a significant increase in intracerebellar cyclic GMP. 3. Pretreatment with the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methylester (L-NAME), but not with D-NAME, significantly reduced the incidence of convulsions and lethality, as well as the increase in cyclic GMP. 4. Pretreatment with N-methyl-D-aspartic acid (NMDA)-receptor antagonist, MK-801, inhibited only the transition of clonic seizure to tonic seizure without affecting the incidence of clonic seizure and lethality. 5. These findings suggest that FQs + BPAA exert convulsions by activating NOS partly through the mediation of the NMDA receptor in the brain cells.

Usefulness of grading of neutrophil aggregate size by laser-light scattering technique for characterizing stimulatory and inhibitory effects of agents on aggregation.

We attempted to apply the particle counting method that employs laser-light scattering technique to quantify the change in numbers of neutrophil homotypic aggregates of 3 graded-sizes (small, medium and large). Ex vivo activation of human neutrophils by a chemotactic peptide, fMLP, predominantly produced small-sized aggregates (< 15 cells), and also, transiently, medium-sized aggregates (16-130 cells). Co-treatment of neutrophils with fMLP and cytochalasin B mainly produced medium-sized aggregates, with very few large-sized aggregates (> 130 cells). Interestingly, when protein kinase C was activated with phorbol 12-myristate 13-acetate small-, medium- and even large-sized aggregates of neutrophils were formed. Presence of extracellular calcium was required to produce these neutrophil aggregations. Both prostaglandin E2 (PGE2) and wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI-3K), inhibited neutrophil aggregation, whereas dbcAMP, a cell permeable analog of cyclic AMP, did not, confirming that PGE2 causes neutrophil aggregation probably through PI-3K inhibition rather than activation of adenylate cyclase. These results suggest that the application of the light scattering technique to characterize human neutrophil aggregates by both size and numbers, has advantages over conventional optical turbulent aggregometry, in that it discriminates neutrophil aggregations produced by different mechanisms of intracellular signaling.

Pharmacokinetics of a New Parenteral Oligosaccharide Antibiotic, SCH27899 (Ziracin), in Healthy Subjects

ABSTRACT The pharmacokinetic properties of an everninomicin antibiotic (SCH27899; Ziracin) were studied with healthy Japanese male volunteers by single (1, 3, 6, and 9 mg/kg of body weight) and multiple 60-min intravenous infusions (3, 6, and 9 mg/kg once daily for 10 consecutive days following a 2-day interval after the initial dose). At single doses the peak serum concentration and the area under the serum concentration-time curve linearly increased with the dose. While total body clearance (CL; 31.2 to 45.6 ml/kg/h) and percent cumulative urinary recovery as unchanged drug (4.9 to 7.1%) were rather constant irrespective of doses, the terminal half-life of γ phase (t1/2γ; 14.2 to 19.6 h) were slightly prolonged at the higher two doses compared with the lower two doses. With repeated doses of SCH27899, a statistically significant decrease and increase were found in CL and t1/2γ of about 36 and 21%, respectively, although these changes may be clinically irrelevant. The most commonly reported adverse events were local reactions such as erythema, pain, and palpable venous cord of mild to moderate degree around the injection site, which could be managed by changing the injection sites.