Noboru Otsuka

Role of tissue plasminogen activator in the sensitization of methamphetamine-induced dopamine release in the nucleus accumbens.

We have previously demonstrated that repeated, but not acute, methamphetamine (METH) treatment increases tissue plasminogen activator (tPA) activity in the brain, which is associated with the development of behavioral sensitization to METH. In this study, we investigated whether the tPA‐plasmin system is involved in the development of sensitization in METH‐induced dopamine release in the nucleus accumbens (NAc). There was no difference in acute METH‐induced increase in extracellular dopamine levels in the NAc between wild‐type and tPA‐deficient (tPA−/−) mice. Repeated METH treatment resulted in a significant enhancement of METH‐ induced dopamine release in wild‐type mice, but not tPA−/− mice. Microinjection of exogenous tPA or plasmin into the NAc of wild‐type mice significantly potentiated acute METH‐ induced dopamine release. Degradation of laminin was evident in brain tissues incubated with tPA plus plasminogen or plasmin in vitro although tPA or plasminogen alone had no effect. Immunohistochemical analysis revealed that microinjection of plasmin into the NAc reduced laminin immunoreactivity without neuronal damage. Our findings suggest that the tPA‐plasmin system participates in the development of behavioral sensitization induced by repeated METH treatment, by regulating the processes underlying the sensitization of METH‐induced dopamine release in the NAc, in which degradation of laminin by plasmin may play a role.

Analgesic Effect of the Newly Developed S(+)-Flurbiprofen Plaster on Inflammatory Pain in a Rat Adjuvant-Induced Arthritis Model

Preclinical Research

Activation of post-synaptic dopamine D1 receptors promotes the release of tissue plasminogen activator in the nucleus accumbens via PKA signaling

We have previously demonstrated that tissue plasminogen activator (tPA) plays an important role through the conversion of plasminogen to plasmin in the release of dopamine in the nucleus accumbens (NAc) evoked by depolarization or the systemic administration of drugs of abuse such as morphine and nicotine. In the present study, we examined the mechanisms by which drugs of abuse increase extracellular tPA activity in the NAc in vivo using in situ zymography. The dopamine D1 receptor (D1R) agonist SKF38393, but not D2 receptor agonist quinpirole, significantly increased extracellular tPA activity in the NAc. The effect of SKF38393 was blocked by pre‐treatment with the dopamine D1R antagonist SCH23390. Microinjection of Rp‐cAMPs, a protein kinase A inhibitor, into the NAc completely blocked the effect of SKF38393. Systemic administration of morphine and methamphetamine increased extracellular tPA activity in the NAc, and these effects were completely blocked by pre‐treatment with SCH23390 and raclopride. The results suggest that activation of post‐synaptic dopamine D1Rs in the NAc leads to an increase in extracellular tPA activity via protein kinase A signaling. Furthermore, dopamine D2 receptors are also involved in the release of tPA induced by morphine and methamphetamine.

Transcriptional induction and translational inhibition of Arc and Cugbp2 in mice hippocampus after transient global ischemia under normothermic condition

Mild hypothermia protects against neuronal damage after transient global ischemia in experimental animals. The exact mechanism of this protective effect remains to be elucidated. The purpose of the present study was to investigate the molecular mechanisms relevant to different neurologic responses to hypothermia and normothermia. Transient global ischemia was induced in C57BL/6 mice by bilateral common carotid artery occlusion for 10 min. Hypothermia provided robust neuroprotection in the hippocampus region and dramatically reduced the mortality rate. Using adaptor-tagged competitive polymerase chain reaction, we obtained the relative transcription levels of 1210 genes in the hippocampal region and compared the expression patterns of these genes. Two genes, Activity-regulated cytoskeleton-associated protein (Arc) and CUG-binding protein-2 (Cugbp2), showed remarkable and persistent increases in their expression levels in normothermic mice, compared with in both sham and hypothermic mice. Despite the increased transcription of Arc and Cugbp2, an immunohistochemistry analysis did not show comparable increases in the translations of both genes. Only a transient increase in Arc protein was observed in the granule cells of the dentate gyrus at 6 h after reperfusion. A remarkable decrease in Cugbp2 protein was observed in the pyramidal cells of the hippocampal CA1-CA3, in accordance with the progress of neuronal degeneration. A decrease in Cugbp2 protein was not observed in hypothermic mice. These results suggest that transient global ischemia induces the translational inhibition of genes with increased expression not in hypothermic, but in normothermic mice. Thus, translational inhibition might play an important role in the progress of neuronal injury after transient global ischemia.

Suppression of AGE Precursor Formation Following Unilateral Ureteral Obstruction in Mouse Kidneys by Transgenic Expression of α-Dicarbonyl/ L -Xylulose Reductase

Unilateral ureteral obstruction (UUO) of kidneys causes acute generation of carbonyl stress. By electrospray ionization/liquid chromatography/mass spectrometry (ESI/LC/MS) we measured the content of methyl glyoxal, glyoxal, and 3-deoxyglucosone in mouse kidney extracts following UUO. UUO resulted in elevation of these dicarbonyls in the obstructed kidneys. Furthermore, the accumulation of 3-deoxyglucosone was significantly reduced in the kidneys of mice transgenic for α-dicarbonyl/L-xylulose reductase (DCXR) as compared to their wild-type littermates, demonstrating 4.91±2.04 vs. 6.45±1.85 ng/mg protein (P=0.044) for the obstructed kidneys, and 3.68±1.95 vs. 5.20±1.39 ng/mg protein (P=0.026) for the contralateral kidneys. On the other hand, collagen III content in kidneys showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal α-dicarbonyl compounds under oxidative circumstances, but it was not sufficient to suppress acute renal fibrosis during 7 d of UUO by itself.

Efficacy of S-flurbiprofen plaster in knee osteoarthritis treatment: Results from a phase III, randomized, active-controlled, adequate, and well-controlled trial

Objectives: S-flurbiprofen plaster (SFPP) is a novel non-steroidal anti-inflammatory drug (NSAID) patch, intended for topical treatment for musculoskeletal diseases. This trial was conducted to examine the effectiveness of SFPP using active comparator, flurbiprofen (FP) patch, on knee osteoarthritis (OA) symptoms. Methods: This was a phase III, multi-center, randomized, adequate, and well-controlled trial, both investigators and patients were blinded to the assigned treatment. Enrolled 633 knee OA patients were treated with either SFPP or FP patch for two weeks. The primary endpoint was improvement in knee pain on rising from the chair as assessed by visual analogue scale (rVAS). Safety was evaluated through adverse events (AEs). Results: The change in rVAS was 40.9 mm in SFPP group and 30.6 mm in FP patch group (p < 0.001). The incidence of drug-related AEs at the application site was 9.5% (32 AEs, 29 mild and 3 moderate) in SFPP and 1.6% in FP patch (p < 0.001). Withdrawals due to AE were five in SFPP and one in FP patch. Conclusions: The superiority of SFPP in efficacy was demonstrated. Most of AEs were mild and few AEs led to treatment discontinuation. Therefore, SFPP provides an additional option for knee OA therapy.

Suppression of Renal α-Dicarbonyl Compounds Generated following Ureteral Obstruction by Kidney-Specific α-Dicarbonyl/l-Xylulose Reductase

Renal unilateral ureteral obstruction (UUO) causes acute generation of α‐dicarbonyl stress substances, such as glyoxal, 3‐deoxyglucosone, and methylglyoxal, in the kidneys. These α‐dicarbonyl compounds are prone to form advanced glycation end products (AGEs) via the nonenzymatic Maillard reaction. Using transgenic (Tg) mice overexpressing a kidney‐specific short‐chain oxidoreductase, α‐dicarbonyl/L‐xylulose reductase (DCXR), we measured generation of α‐dicarbonyls following UUO by means of electrospray ionization/liquid chromatography/mass spectrometry in their kidney extracts. The accumulation of 3‐deoxyglucosone was significantly reduced in the kidneys of the mice Tg for DCXR compared to their wild‐type littermates, demonstrating 4.91 ± 2.04 vs. 6.45 ± 1.85 ng/mg protein (P = 0.044) for the obstructed kidneys, and 3.68 ± 1.95 vs. 5.20 ± 1.39 ng/mg protein (P = 0.026) for the contralateral kidneys. Despite the reduction in accumulated α‐dicarbonyls, collagen III content in kidneys of the Tg mice and their wild‐type littermates showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal α‐dicarbonyl compounds under oxidative circumstances, but it is not sufficient to suppress acute renal fibrosis during 7 days UUO.

Topical Anti-Inflammatory and Analgesic Effects of Multiple Applications of S(+)-Flurbiprofen Plaster (SFPP) in a Rat Adjuvant-Induced Arthritis Model

Preclinical Research

Pharmacological action and clinical outcome of newly developed NSAIDs patch, “LOQOA ® tape”

Topical non-steroidal anti-inflammatory drugs (NSAIDs) patches are indispensable for the treatment of musculoskeletal diseases, while they are considered less effective than oral NSAIDs. LOQOA® tape is a tape-type patch containing esflurbiprofen (SFP) as a major active ingredient with potent cyclooxygenase inhibition and sufficient skin permeability. SFP patch (SFPP) showed higher percutaneous absorption rate, rapid pain relief, and potent anti-inflammatory efficacy comparing with existing NSAIDs patches in rat. SFPP showed dramatically higher synovial fluid and tissue concentration on SFP than that of flurbiprofen (FP) patch after single application to knee osteoarthritis (OA) patients. On the other hand, clinical dosage of SFPP was determined as not more than two patches a day from the estimation of systemic exposure to SFP of SFPP and oral FP. SFPP showed statistically significant differences in pain relief and all the other efficacy end points compared to inactive placebo or FP patch in knee OA patients. Efficacy on OA other than knee joint was also observed. In long-term study of SFPP, using up to two patches a day, a total of 201 patients was included and 161 patients achieved 52-week application. Among drug-related side effects, skin reaction at the application sites was observed in 46.8% and discontinued in 4.3%. Although gastro-intestinal reaction and abnormal changes in laboratory tests related to kidney function were observed as systemic drug-related side effects, most of them were mild in severity. SFPP, the new generation NSAIDs patch, would be one of effective options for the treatment of symptomatic OA patients.

The efficacy and safety of S-flurbiprofen plaster in the treatment of knee osteoarthritis: a phase II, randomized, double-blind, placebo-controlled, dose-finding study

Background Nonsteroidal anti-inflammatory drug (NSAID) patches are convenient for use and show much less gastrointestinal side effects than oral NSAIDs, whereas its percutaneous absorption is not sufficient for the expression of clinical efficacy at satisfactory level. S-flurbiprofen plaster (SFPP) has shown dramatic improvement in percutaneous absorption results from animal and clinical studies. In this study, the efficacy and safety of SFPP were compared with placebo in patients with knee osteoarthritis (OA) to determine its optimal dose. This was a multicenter, randomized, double-blind, parallel-group comparative study. Patients and methods Enrolled 509 knee OA patients were treated with placebo or SFPP at 10, 20, or 40 mg applied on the affected site once daily for 2 weeks. The primary endpoint for efficacy was improvement in knee pain on rising from the chair assessed by visual analog scale (VAS). The other endpoints were clinical symptoms, pain on walking, and global assessment by both investigator and patient. Safety was evaluated by observing adverse events (AEs). Results VAS change in knee pain from baseline to trial end was dose-dependent, least squares mean was 29.5, 31.5, 32.0, and 35.6 mm in placebo and SFPP 10, 20, and 40 mg, respectively. A significant difference was observed between placebo and SFPP 40 mg (P=0.001). In contrast, the effect of SFPP at a dose ≤20 mg was not significantly different from that of placebo. The proportion of the patients who achieved 50% pain relief was 72.4% in 40 mg and 51.2% in placebo (P<0.001). In all other endpoints, SFPP 40 mg showed significant improvement compared with placebo. The incidence of AEs was not different across all four groups, and no severe AEs were observed. Conclusion Clinically relevant pain relief was observed in all groups including placebo. Especially 40 mg showed remarkable pain relief in not only primary endpoint but also all the other endpoint with significant differences over placebo. The safety profile of SFPP 40 mg was not different from that of placebo. Therefore, 40 mg was determined as the optimal tested dose.