Iwao Arai

NS-398, a new anti-inflammatory agent, selectively inhibits prostaglandin G/H synthase/cyclooxygenase (COX-2) activity in vitro

NS-398 is a novel anti-inflammatory and analgesic agent which produces much fewer gastrointestinal lesions in rats. Recently, two forms of cyclooxygenase have been identified: a COX-1 first purified from ram seminal vesicles and a newly discovered mitogen-inducible form (COX-2). Effects of NS-398 on activities of these two distinct forms of COX were investigated. COX-1 purified from ram seminal vesicles and COX-2 isolated from sheep placenta (purity was 70%) were used. The COX-1 activity was completely unaffected by 10(-4) M NS-398, whereas the COX-2 activity was concentration-dependently inhibited, the IC50 value being 3.8 x 10(-6) M. Indomethacin inhibited both COX-1 and COX-2 activity to the same degree, the IC50 values being 7.4 x 10(-7) M and 9.7 x 10(-7) M, respectively. The anti-inflammatory and analgesic effects of NS-398 were almost as potent as indomethacin, the effective dose range being 0.3 approximately 5 mg/kg in rats. The gastrointestinal lesions related to NS-398 were not significant following a dose of 1000 mg/kg given orally. NS-398 is the first documented agent to have selective inhibition for COX-2, which may result in the less gastrointestinal toxicity.

NS-398, a novel non-steroidal anti-inflammatory drug with potent analgesic and antipyretic effects, which causes minimal stomach lesions.

1. NS-398 (N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide) is a new non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic effects. 2. The anti-inflammatory potency of NS-398 in rat carrageenin-induced edema was as potent as that of indomethacin and 8 times more potent than diclofenac. In rat adjuvant arthritis, NS-398 showed a therapeutic effect comparable to that seen with loxoprofen but less than that seen with indomethacin and diclofenac. 3. The analgesic potency of NS-398 in rat adjuvant arthritic pain was much the same as that of indomethacin, and was about 3-5 times higher than that of diclofenac and loxoprofen. In the Randall-Selitto method in rats, NS-398 was 2-7 times as potent as loxoprofen, diclofenac and indomethacin. In acetic acid-induced writhing in mice, NS-398 was equipotent to indomethacin and diclofenac. 4. In LPS-induced fever in rats, NS-398 was 1.5-4.5 times as potent as loxoprofen and indomethacin, but less potent than diclofenac. 5. NS-398 produced little gastric ulceration in doses of up to 1000 mg/kg, while reference drugs produced distinct stomach lesions in doses of 10-30 mg/kg. 6. NS-398 inhibited prostaglandin (PG) endoperoxide synthase from sheep seminal vesicle microsomes less potent than that of ibuprofen.

Selective inhibition of NS-398 on prostanoid production in inflamed tissue in rat carrageenan-air-pouch-inflammation

Abstract— NS‐398 (N‐(2‐cyclohexyloxy‐4‐nitrophenyl) methane sulphonamide), a newly synthesized potent non‐steroidal antiinflammatory drug (NSAID) has a much lesser degree of toxicity, as compared with presently available NSAIDs. We have investigated the inhibition of prostanoid production in inflammatory exudate, gastric mucosa and renal papillary tissue, following oral administration to carrageenan‐air‐pouch rats. The ID50 values of NS‐398 in the inflammatory exudate, gastric mucosa and renal papillary tissue were 0·18, 62·2 and 261·7 mg kg−1, respectively. In contrast, indomethacin decreased the PGE2 concentration in the inflammatory exudate, gastric mucosa and renal papillary tissue, with the same dose range, the ID50 values being 0·23, 0·14 and 0·15 mg kg−1, respectively. The same tendency was seen for 6‐keto‐prostaglandin F1 and thromboxane B2. Moreover, NS‐398 inhibited excess PGE2 production in inflamed tissue but did not affect physiological production of PGE2 in non‐inflamed tissue. Indomethacin, in both inflamed and non‐inflamed tissues, inhibited PGE2 production to the same degree. These results indicated that NS‐398 has some specificity for inflamed tissue, by inhibiting prostanoid synthesis, and this effect may explain the decreased side‐effects of this drug.

Involvement of IL‐31 on scratching behavior in NC/Nga mice with atopic‐like dermatitis

Abstract:  Pruritus is an important symptom in atopic dermatitis (AD), but the major pruritogen have not been identified. NC/Nga mice, spontaneously develop an eczematous AD‐like skin lesion when kept under conventional conditions, but not under specific pathogen‐free (SPF) conditions, have been thought to be an animal model for AD. In this study, to determine whether newly identified cytokine, IL‐31, may be involved in pruritus of AD, we examined the IL‐31 expression in spontaneous dermatitis model which showed itch‐associated long‐lasting (over 1.5 s duration) scratching behavior and compared with that of hapten‐induced contact dermatitis model without itch‐associated long‐lasting scratching behavior, using NC/Nga mice. In NC/Nga mice cohabited with NC/Nga mice which developed severe dermatitis for 2 weeks (conventional NC/Nga mice), the numbers of long‐lasting scratching counts were significantly increased. Yet in 2,4,6‐trinitrochlorobenzene (TNCB)‐sensitized and challenged mice (TNCB‐applied NC/Nga mice), no significant increase in long‐lasting scratching counts was observed. In conventional NC/Nga mice with long‐lasting scratching behavior, expression of IL‐31 mRNA was increased, while in TNCB‐applied NC/Nga mice without long‐lasting scratching behavior, the expression of IL‐31 mRNA were unchanged. There was a good correlation between the scratching counts and expression of IL‐31 mRNA in conventional NC/Nga mice, but not so in TNCB‐applied NC/Nga mice. These results suggest that IL‐31 causes the itch‐associated scratching behavior in conventional NC/Nga mice, an experimental animal model for AD.

Analysis of the spontaneous scratching behavior by NC/Nga mice: a possible approach to evaluate antipruritics for subjects with atopic dermatitis

We investigated the spontaneous scratching by NC/Nga mice to design a new method for evaluating the itch of subjects with atopic dermatitis. The numbers of scratchings in various strains of mice were classified based on the duration of the scratching. Prolonged scratching was frequent in skin-lesioned NC/Nga mice, but not in ICR, BALB/c and non-lesioned NC/Nga mice. Pretreatment with dexamethasone or tacrolimus significantly suppressed long-duration scratching in NC/Nga mice but did not suppress short-duration scratching induced by ovalbumin active cutaneous anaphylaxis in BALB/c mice and in ICR mice subcutaneously injected with histamine. In contrast, pretreatment with chlorpheniramine or ketotifen significantly suppressed short-duration scratching induced by ovalbumin active cutaneous anaphylaxis in BALB/c mice and in ICR mice subcutaneously injected with histamine, but not long-duration scratching seen in NC/Nga mice. These findings indicate that the mechanism of spontaneous scratching in NC/Nga mice differs from that induced by several pruritogen injections. This new method shows good correlation with the therapeutic activity of drugs in cases of atopic dermatitis in humans and may serve as a useful model for evaluating antipruritic drugs and for studying mechanisms involved in atopic dermatitis.

Modulation of scratching behavior by silencing an endogenous cyclooxygenase-1 gene in the skin through the administration of siRNA

RNA interference (RNAi) is rapidly becoming a major tool that is revolutionizing research in the bioscience and biomedical fields. To apply the RNAi technique in vivo, it is crucial to develop appropriate methods of guiding the short interfering RNA (siRNA) molecules to the right tissues and cells. Here, we demonstrate an efficient method for performing gene knockdown in the body skin using the in vivo electro‐transduction of siRNA. Using this method, we examined whether the targeted silencing of the cyclooxygenase (COX) gene in the skin could modulate the scratching behavior of an atopic dermatitis mouse model.

Inhibition of gastric H+,K+-ATPase and acid secretion by cassigarol A, a polyphenol from Cassia garrettiana Craib

The effects of cassigarol A, a naturally occurring polyphenol, on gastric H+,K(+)-ATPase and gastric acid secretion were studied. Cassigarol A inhibited H+,K(+)-ATPase and K-stimulated p-nitrophenyl phosphatase from hog gastric mucosa with 50% inhibition of 1.2 x 10(-6) and 6.3 x 10(-6) M, respectively. The kinetic study showed that the inhibition of H+,K(+)-ATPase by cassigarol A was competitive with respect to ATP and non-competitive with respect to K+. Cassigarol A inhibited both H+,K(+)-ATPase-mediated proton transport and 2-deoxy-D-glucose-induced acid secretion. On the other hand, cassigarol A acetate, in which phenolic hydroxy groups are acetylated, was not effective in the inhibition of enzyme activity and acid secretion. These results indicate that cassigarol A is a potent inhibitor of gastric H+,K(+)-ATPase, that the anti-secretory activity of cassigarol A is related to the inhibition of H+,K(+)-ATPase and that an important moiety of cassigarol A in the interaction with the enzyme is the phenolic hydroxy groups.

A method to induce stable atopic dermatitis-like symptoms in NC/Nga mice housed with skin-lesioned mice.

Background  Itching is a characteristic symptom in various forms of dermatosis, especially atopic dermatitis; consequently it is a major diagnostic criterion. All features are similar to events seen in patients, hence NC/Nga mice are considered to be a suitable model of human atopic dermatitis. However, there were data spreads in commencing time and the degree of skin lesions in NC/Nga mice.

Effects of restraint and water-immersion stress and insulin on gastric acid secretion in rats

Effects of restraint and water-immersion stress (RWIS) and of insulin injection on gastric acid secretion were investigated in relation to blood glucose levels and to brain glucose uptake in rats. Venous blood glucose levels (VBG) were significantly increased while arterial blood glucose level (ABG) was slightly increased by RWIS. In contrast, ABG and VBG were significantly decreased by administration of insulin; the decrease in ABG was greater than that in VBG. Glucose uptake into the brain, calculated from the ABG-VBG, was significantly decreased both by RWIS loading and by insulin administration. The uptake of [14C] 2-deoxy-D-glucose [( 14C]-2DG) into the brain was also significantly decreased in RWIS-loaded or insulin-treated rats. Gastric acid output was significantly increased both by RWIS loading and by insulin administration. The increased acid output paralleled the decrease of glucose uptake into the brain in RWIS-loaded and insulin-treated rats. Results suggest that RWIS-induced gastric acid secretion is regulated by brain glucose uptake and that this gastric acid secretion is a hypothalamic neuron-mediated event as is insulin-stimulated gastric acid secretion.

Scratching behavior in spontaneous- or allergic contact-induced dermatitis in NC/Nga mice.

Abstract:  NC/Nga mice have pathological and behavioral features similar to those seen in human atopic dermatitis. There are two known dermatitis models in NC/Nga mice, one being spontaneous‐induced dermatitis under conventional conditions and the other 2,4,6‐trinitrochlorobenzene (TNCB)‐induced allergic contact dermatitis. However, there are significant differences in time course on development of dermatitis. We studied the role of scratching behavior (sign of itch) on the development of dermatitis on spontaneous‐ and TNCB‐induced dermatitis. We measured scratching counts, transepidermal water loss (TEWL), and skin inflammation score, under conventional conditions or by applying 5% TNCB once a week for 6 weeks in NC/Nga mice. In spontaneous‐induced dermatitis, scratching counts increased with the passage of time. The scratching counts were significantly increased only 1 week after housing the mice under conventional conditions, but no changes were observed in cases of TNCB‐induced dermatitis. In spontaneous‐induced dermatitis, TEWL and skin‐inflammation score were gradually increased, time‐dependently. On the other hand, in TNCB‐induced dermatitis, these dependent values rapidly increased and reached a maximum only after 24 h TNCB application. These data suggest that pathogenesis of spontaneous‐ and allergic contact‐induced dermatitis was clearly different. It will be of major interest to identify the pruritic mediators causing profound scratching behavior and scratching‐induced aggravation of inflammation in the spontaneous‐induced dermatitis, as opposed to the inflammatory mediators that cause contact allergic dermatitis without major scratching.