Nobuko Futaki

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.

Selective inhibition of cyclooxygenase-2 by NS-398 in endotoxin shock rats in vivo

Abstract.Objective and Design: The role of cyclooxygenase (COX)-2 was examined using a rat endotoxin shock model and the potency and selectivity of NS-398, a COX-2 selective inhibitor in vitro, for COX-2 activity was examined in vivo.¶Material: Male Wistar rats (weighing 140–180 g) were used.¶Methods: Lipopolysaccharide (LPS, 1 mg/kg, i.v.) was administered to rats (LPS-treated rats) and expression of COX-1 mRNA and COX-2 mRNA in the aorta and peripheral blood leukocytes was examined by RT-PCR. COX activity was assessed by measuring the plasma 6-keto prostaglandin (PG) F1α, PGE2 and thromboxane (TX) B2 30 s after administration of arachidonic acid (AA, 3 mg/kg, i.v.). NS-398 (0.3–100 mg/kg, p.o.) or indomethacin (0.3–3 mg/kg, p.o.) was administered 1 h before the AA injection.¶Results: COX-2 mRNA was detectable in the aorta and peripheral blood leukocytes at least from 3 to 9 h after the LPS injection but not in non-LPS-treated rats. Plasma 6-keto PGF1α, PGE2 and TXB2 levels after AA injection into LPS-treated rats were significantly enhanced compared to findings in non-LPS-treated rats. NS-398 showed significant inhibition of the increase in PGs in LPS-treated rats, the ED50 values being 0.35 mg/kg for 6-keto PGF1α, 1.5 mg/kg for PGE2 and < 0.3 mg/kg for TXB2. NS-398 even at 100 mg/kg did not significantly suppress the increased PGs levels in non-LPS-treated rats. In contrast, indomethacin significantly inhibited plasma PGs levels after AA injection into LPS-treated rats and non-LPS-treated rats. The ED50 values in LPS-treated rats, determined by 6-keto PGF1α, PGE2 and TXB2 production, were 1.0, 1.3 and 2.3 mg/kg and those in non-LPS-treated rats were 0.42, 0.24 and 0.93 mg/kg, respectively.¶Conclusions: In a rat endotoxin shock model, expression of COX-2 plays a role in an increase in COX activity. NS-398 showed preferential inhibitory effects on COX-2 activity in vivo. This approach is useful to directly analyze the inhibitory activity of NSAIDs for COX-1 and COX-2 in vivo.

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.

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.

Role of scratch-induced cutaneous prostaglandin D2 production on atopic-like scratching behaviour in mice

Abstract:  NC/Nga mice are known to develop scratching dermatitis akin to atopic dermatitis, under conventional (Conv), but not under the specific‐pathogen‐free (SPF) condition. In this study, we examined the effects of mechanical‐scratching on the spontaneous scratching counts (sign of itching), in relation to the cutaneous prostaglandin D2 (PGD2) levels in NC/Nga or BALB/c mice. Mechanical‐scratching increased the cutaneous barrier damage and PGD2 levels in both strain mice under the SPF condition. By 4 weeks of cohabitation with the skin‐lesioned NC/Nga mice, both the increase in the spontaneous scratching and development of dermatitis score were higher in the Conv‐NC/Nga than in the Conv‐BALB/c mice. At this time‐point, the cutaneous PGD2 level induced by mechanical‐scratching was significantly lower in the Conv‐NC/Nga when compared with that in the SPF‐NC/Nga mice, and that in the Conv‐BALB/c was almost equal to that in the SPF‐BALB/c mice. With mechanical scratches, the cohabitation‐induced scratching was suppressed in the Conv‐BALB/c, but not in the Conv‐NC/Nga mice. These results suggest that the scratch‐induced cutaneous PGD2 inhibits scratching and the subsequent development of dermatitis in BALB/c, while the impaired scratch‐induced cutaneous PGD2 production in the NC/Nga mice resulted in no suppression of scratching, and aggravated the dermatitis.

Effects of combined treatment with eldecalcitol and alendronate on bone mass, mechanical properties, and bone histomorphometry in ovariectomized rats: A comparison with alfacalcidol and alendronate

Eldecalcitol (ELD), a 2β-hydroxypropyloxy derivative of 1α,25 (OH) 2D3, inhibits bone resorption more potently than alfacalcidol (ALF) while maintaining osteoblastic function in an ovariectomized (OVX) osteoporosis rat model. Alendronate (ALN), which is the most common bisphosphonate used for the treatment of osteoporosis, increases the bone mineral density (BMD) by suppressing bone resorption. In this study, we investigated the effects of combination treatments with ELD and ALN or with ALF and ALN on bone mass and strength in OVX rats. Seventy female rats, 32 weeks old, were assigned to seven groups: (1) a sham-operated control group; (2) an OVX-control group; (3) an ELD group; (4) an ALF group; (5) an ALN group; (6) an ELD+ALN group; and (7) an ALF+ALN group. OVX rats were orally treated with ELD (0.015 μg/kg), ALF (0.0375 μg/kg), or ALN (0.2mg/kg) daily for 12 weeks. In both the lumbar spine and the femur, ELD and ALF monotherapy significantly increased the BMD, and ELD+ALN and ALF+ALN significantly increased the BMD, compared with ALN monotherapy, as an additive effect. In particular, ELD+ALN resulted in a significantly higher BMD than ALF+ALN in the femur. On mechanical testing of the lumbar spine, ELD and ALF monotherapy significantly increased the ultimate load, and ELD+ALN and ALF+ALN significantly increased the ultimate load compared with ALN monotherapy. In the femur, ELD, ELD+ALN, and ALF+ALN treatment significantly increased the ultimate load, compared with the OVX-control group, and ELD+ALN resulted in a significantly higher ultimate load than ALN monotherapy. A histomorphometric analysis showed that ELD monotherapy and ELD+ALN combination therapy had a potent inhibitory effect on bone resorption parameters (osteoclast surface and eroded surface), while maintaining bone formation parameters (osteoblast surface and osteoid surface). By contrast, ALF and ALF+ALN significantly lowered the histological parameters of both bone resorption and formation. These results suggested that ELD or ALF used in combination with ALN has therapeutic advantages over ALN monotherapy, with ELD+ALN combination treatment producing an especially beneficial anti-osteoporotic effect by inhibiting osteoclastic bone resorption and maintaining osteoblastic function, compared with ALF+ALN combination treatment.

High levels of human recombinant cyclooxygenase-1 expression in mammalian cells using a novel gene amplification method.

We report the expression of a high level of human cyclooxygenase-1 (hCOX-1) in mammalian cells using a novel gene amplification method known as the IR/MAR gene amplification system. IR/MAR-plasmids contain a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) and amplify autonomously without a specific induction process. In this study, the IR/MAR-plasmid pΔBN.AR1 was cotransfected with pCAG-COX1, which expresses hCOX-1, into human HEK293T cells, and G418 and blasticidin S double-resistant cells were obtained in about 1month. Real-time PCR and Western blotting revealed that the expressions of hCOX-1 mRNA and protein in both polyclonal and monoclonal cells were remarkably higher than those in only pCAG-COX1-transfected control cells. Southern blotting demonstrated the amplification of the hCOX-1 gene, and the copy number of clone #43 obtained by the cotransfection of pΔBN.AR1 and pCAG-COX1 was more than 20 copies per cell, though that of clone #14 obtained without using the IR/MAR plasmid pΔBN.AR1 was only two copies. These results indicate that a high level of hCOX-1 expression was achieved as a result of hCOX-1 gene amplification. Furthermore, the crude extract from clone #43 showed a strong COX-1 activity, and the activity was inhibited by the representative COX-1 inhibitor indomethacin, with an IC(50) value of 36nM. These results demonstrate that the IR/MAR gene amplification system is a simple but useful method for generating highly productive mammalian cells.