Reiko Eyanagi

Acid sphingomyelinase inhibition suppresses lipopolysaccharide-mediated release of inflammatory cytokines from macrophages and protects against disease pathology in dextran sulphate sodium-induced colitis in mice

Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The design of SMase inhibitors may offer new therapies for the treatment of LPS‐ and cytokine‐related inflammatory bowel disease. We synthesized a series of difluoromethylene analogues of SM (SMAs). We report here the effects of the most potent SMase inhibitor, SMA‐7, on the LPS‐mediated release of tumour necrosis factor‐α, interleukin‐1β and interleukin‐6 from THP‐1 macrophages and the pathology of dextran sulphate sodium (DSS)‐induced colitis in mice. SMA‐7 suppressed the LPS‐induced cytokine release and nuclear factor‐κB activation. LPS stimulation caused a four‐fold increase in acid SMase activation, but little increase in neutral SMase activity. The presence of 10 μm SMA‐7 caused acid SMase to remain at the control levels and reduced the formation of ceramide. HT‐29 cells had significantly decreased cell viability when incubated with media from LPS‐stimulated THP‐1 macrophages. However, incubating the colon cells in media from both SMA‐7 and LPS‐treated macrophages caused little decrease in viability, suggesting that ceramide has a role in the LPS‐stimulated signalling that releases cytotoxic factors against colon cells. Oral administration of SMA‐7 to mice with 2% DSS in the drinking water, for 10 or 21 consecutive days, reduced significantly the cytokine levels in the colon and the severity of colonic injury. These findings suggest a central role for acid SMase/ceramide signalling in the pathology of DSS‐induced colitis in mice, indicating a possible preventive or therapeutic role for SMase inhibitor in inflammatory bowel disease.

Design, Synthesis, and Pharmacological Activity of Nonallergenic Pyrazolone-Type Antipyretic Analgesics

To develop novel nonallergenic pyrazolone analgesics, we synthesized a series of compounds in which position 1 of the pyrazolone ring was substituted in place of the original methyl group in order to block the formation of allergenic metabolites via N-dealkylation. These pyrazolone analogues were found to show as potent an antipyretic and analgesic effect as antipyrine (AT). In an examination of allergenicity, AT induced a typical skin reaction in guinea pigs, whereas the pyrazolone analogues were inactive. When AT was administered (po) to rats, norantipyrine (NORA) as an active metabolite was detected in the urine, whereas similar administration of the pyrazolone analogues did not afford NORA. We conclude that these novel pyrazolone analogues were nonallergenic because they were not converted to allergenic metabolites in vivo. Because these compounds retain the antipyretic and analgesic activities of AT, they are considered to be promising candidates for nonallergenic antipyretic analgesics.

Role of amygdaloid nuclei in the anxiolytic-like effect of nociceptin/orphanin FQ in rats

We investigated the mechanism underlying the anxiolytic actions of the neuropeptide nociceptin/orphanin FQ (N/OFQ) using the elevated plus-maze test and T-maze test. Microinfusions of N/OFQ (10 or 32pmol) into the central amygdala (ACE) increased the time spent in the open arms of the elevated plus-maze (anxiolytic-like effects), whereas microinfusions of N/OFQ (10, 32 or 100 pmol) into the basolateral amygdala (ABL) did not affect the time spent in the open arms. Moreover, microinfusions of N/OFQ (32 pmol) into the ACE impaired escape performance from the open arms of the elevated T-maze (anxiolytic-like effects), but did not change inhibitory avoidance of the open arms. A non-peptidyl N/OFQ receptor (NOP) antagonist, J-113397(1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) (10 mg/kg, s.c.), blocked the anxiolytic-like effects induced by N/OFQ. These results indicate that the anxiolytic-like effects of N/OFQ might be due to impaired escape performance from the open arms and it implicates the N/OFQ system within the ACE in the mediation of panic action.

Involvement of the GABA/benzodiazepine receptor in the axiolytic-like effect of nociceptin/orphanin FQ

We investigated the mechanism underlying the anxiolytic actions of the neuropeptide nociceptin/orphanin FQ (N/OFQ) with an elevated plus-maze test. In mice, intracerebroventricular (i.c.v.) infusions of N/OFQ (0.1 and 0.32 nmol) led to an increase in time spent in the open arms (anxiolytic-like effects). A non-peptidyl N/OFQ receptor (NOP) antagonist, J-113397(1-{(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl}-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one), (1.0 and 3.2 mg/kg, s.c.) blocked the increase induced by N/OFQ. On the other hand, a benzodiazepine receptor antagonist, flumazenil, (10 mg/kg, i.p.) and a GABAA receptor antagonist, (+)-bicuculline, (5.6 mg/kg, i.p.) also inhibited the increase induced by N/OFQ. In rats, microinfusions of N/OFQ (10 and 32 pmol) into the amygdala led to an increase in time spent in the open arms. However, intracranial infusions of N/OFQ (10-100 pmol) into the dorsal hippocampus did not affect the time spent in the open arms. These findings suggest that the anxiolytic-like effects of N/OFQ may be related to the GABA/benzodiazepine system in the amygdala.

The stimulatory effects of caffeine with oseltamivir (Tamiflu) on light-dark behavior and open-field behavior in mice.

Abnormal behaviors and death associated with the use of oseltamivir (Tamiflu) have emerged as a major issue in influenza patients taking the drug. Here, we investigated the mechanisms underlying the effects of oseltamivir on the behavior of mice using light-dark and open-field preference tests. Oseltamivir (75 and 150 mg/kg, intraperitoneally (i.p.)) alone affected neither time spent in the open area in the light-dark preference test nor ambulation in the open-field test at 2h post-injection. However, a non-selective adenosine A(1)/A(2) receptor antagonist, caffeine (10mg/kg, i.p.) in combination with oseltamivir (150 mg/kg, i.p.) increased time spent in the open area in the light-dark preference test. This enhancement was not inhibited by a benzodiazepine receptor antagonist, flumazenil (10-20mg/kg, subcutaneously (s.c.)). Enhancement of ambulation in the open-field test was also observed when caffeine (10mg/kg, i.p.) was combined with oseltamivir (150 mg/kg, i.p.). This enhancement was inhibited by a dopamine D(2) receptor antagonist, haloperidol (0.1mg/kg, s.c.). Furthermore, an adenosine A(2) receptor antagonist, SCH58261 (3mg/kg, i.p.) in combination with oseltamivir (150 mg/kg, i.p.) increased ambulation in the open-field test, while an adenosine A(1) receptor antagonist, DPCPX (1-3mg/kg, i.p.) did not. These findings suggest that the actions of oseltamivir may involve the dopamine and adenosine systems. Our findings suggest that due to the interaction between central blockade of adenosine A(2) receptors by caffeine, and oseltamivir-induced behavioral changes, patients being treated with oseltamivir should be closely monitored.

Antigenicity of sulfanilamide and its metabolites using fluorescent-labelled compounds

In order to clarify the onset mechanisms of drug-induced allergies, three fluorescent-labelled compounds were synthesized by subjecting sulfanilamide (SA), a base compound for sulfonamides, and its active metabolites, i.e. sulfanilamide hydroxylamine and sulfanilamide nitroso, to dansylation using dansylchloride. In other words, 5-dimethylamino-N-(4-aminobenzyl)-naphthalenesulfonamide (DNS-4ABA), 5-dimethylamino-N-(4-hydroxylaminobenzyl)-1-naphthalenesulfonamide (DNS-4HABA) and 5-dimethylamino-N-(4-nitrosobenzyl)-1-naphthalenesulfonamide (DNS-4NSBA) were synthesized as model haptens. When analysed by HPLC, a conjugate of DNS-4HABA and glutathione (GSH) with nucleophilic amino acids had two peaks (P-1 and P-2). FAB-MS and 1H-NMR revealed that the DNS-4HABA-GSH conjugate consisted of sulphinamide and semimercaptal. The reactivity of GSH to DNS-4ABA, DNS-4HABA and DNS-4NSBA was quantified by HPLC using an oxidization system (horseradish peroxidase/H2O2). The results show that production of DNS-4NSBA-GSH-conjugate was four to eight times higher than that of DNS-4HABA-GSH conjugate, but that DNS-4ABA did not bind with GSH. Skin reactions were assessed using guinea pigs, and strong delayed erythema was seen with DNS-4NSBA, which bound most strongly with GSH, whereas weak delayed erythema was seen with DNS-4ABA, which did not bind with GSH. This suggests a correlation between GSH conjugate production and skin reactions. DNS-4HABA enzymatically bound with proteins in rat and guinea pig liver cytosol and microsomal fractions. The proteins that bound to DNS-4HABA were purified by HPLC and then subjected to N-terminal amino acid analysis. Ubiquitin (10 kDa) and fatty acid binding protein (30 kDa) were detected in the rat liver cytosol fraction; retinol-dehydrogenase (35 kDa) in the rat microsomal fraction; and glutathione-S-transferase B (mμ) (25 kDa) in the guinea pig liver cytosol fraction. When DNS-4HABA or DNS-4NSBA binds to proteins that play important roles in the body, unexpected adverse reactions may occur. Furthermore, by utilizing our technique using model compounds, it may be possible to identify the carrier proteins of various compounds, including pharmaceutical agents.

Covalent binding of nitroso-sulfonamides to glutathione S-transferase in guinea pigs with delayed type hypersensitivity

Drug induced allergies are believed to be induced by conjugates consisting of biological macromolecules and active metabolites. The present study investigated whether guinea pig glutathione S-transferase (gpGST), a protein that binds with sulfanilamide (SA) and sulfamethoxazole (SMX), could be detected in the liver cytosol fraction of guinea pigs that intraperitoneally received SA or SMX, and whether gpGST is a carrier protein. We synthesized three nitroso compounds, i.e., 4-nitroso-sulfanilamide (SA-NO), 4-nitrososulfamethoxazole (SMX-NO) and fluorescent-labeled nitroso compound (DNSBA-NO), and examined binding quantities of nitroso compounds to gpGST purified from untreated female guinea pigs. Furthermore, the concentrations of IgG in serum antibody for nitroso compounds were estimated using ELISA. When guinea pigs were sensitized using the three nitroso compounds, the dose dependent skin reactions were confirmed with each compound. In addition, sensitized guinea pigs using each nitroso compound showed positive skin reactions at an elicitation test performed using gpGST alone. The results confirmed synthesis of antibody against gpGST due to hapten sensitization. Therefore, when a nitroso compound binds with gpGST in the body of guinea pigs, nitroso-gpGST acts as a neoantigen, which induces synthesis of autoantibody. Thus, gpGST appears to be one of the carrier proteins that induce sulfa drug-induced allergies. Immunization of guinea pigs with active metabolite of drugs may give information for predicting the occurrence of delayed type hypersensitivity in human.