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Furan, oxazole-derived Schiff base, and different aldehydes are to be combined to study their potential activities including anti-inflammatory action through COX enzyme inhibition. Molecular docking studies determine the most detailed probable view of drug-receptor interaction and have created a new rational approach to drug design. It suggests that these twelve compounds have strong interaction with COX-1 and COX-2 enzymes, which are responsible for the activity. In this article, 12 oxazole derivatives were docked inside cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzyme’s crystal structures to calculate the binding potency of each derivative within the active site. Molecular modeling and In silico prediction of ADME properties approaches were conducted and the two most effective derivatives were chosen with docking binding range (-10.311 to -9.02) kcal/mol and (-9.642 to -9.18) respectively. The binding ability of indomethacin, aspirin, and mofezolac were selected as non-selective COX inhibitors. Whereas celecoxib, valdecoxib, rofecoxib, and parecoxib stand for selective COX inhibitors.

Cardiovascular diseases (CVDs) account for over 17 million death globally each year, including arterial thrombosis. Platelets are key components in the pathogenesis of this disease and modulating their activity is an effective strategy to treat such thrombotic events. Cyclooxygenase-1 (COX-1) isoenzyme is involved in platelet activation and is the main target of non-steroidal anti-inflammatory drugs (NSAIDs) and new selective inhibitor research. Inhibitors of general formula mofezolac-spacer-mofezolac (mof-spacer-mof) and mofezolac-spacer-arachidonic acid (mof-spacer-AA) were projected to investigate the possible cross-talk between the two monomers (Eallo and Ecat) forming the COX-1 homodimer. Mofezolac was chosen as either one or two moieties of these molecules being the known most potent and selective COX-1 inhibitor and administrated to humans as Disopain™, then arachidonic acid (AA) was used to develop molecules bearing, in the same compound, in addition to the inhibitor moiety (mofezolac) also the natural COX substrate. Depending on the nature of the spacer, COX-1 and COX-2 activity was differently inhibited by mof-spacer-mof set with a preferential COX-1 inhibition. The highest COX-1 selectivity was exhibited by the compound in which the spacer was the benzidine [N,N’-(biphenyl-4,4′-di-yl)bis (2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamide) (15): COX-1 IC50 = 0.08 μM, COX-2 IC50 > 50 μM, Selectivity Index (SI) > 625]. In the case of mof-spacer-AA set, the COX inhibitory potency and also the isoform preference changed. (5Z, 8Z, 11Z, 14Z)-N-(4-{2-[3,4-Bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}butyl)icosa-5,8,11,14-tetraenamide (19) and (5Z, 8Z, 11Z, 14Z)-N-(4’-{2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}-[1,1′-biphenyl]-4-yl)icosa-5,8,11,14-tetraenamide (21), in which the spacer is the 1,2-diaminobutane or benzidine, respectively, selectively inhibited the COX-2, whereas when the spacer is the 1,4-phenylendiamine [(5Z, 8Z, 11Z, 14Z)-N-(4-{2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}phenyl)icosa-5,8,11,14-tetraenamide) (20) the COX preference is COX-1 (COX-1 IC50 = 0.05 μM, COX-2 IC50 > 50 μM, with a COX-1 selectivity > 1000). Molecular modelling by using FLAP algorithm shows fundamental interactions of the novel compounds at the entry channel of COX and inside its catalytic cavity. The effect of these mof-spacer-mof and mof-spacer-AA in inhibiting in vitro free arachidonic acid-induced platelet aggregation was also determined. A positive profile of hemocompatibility in relation to their influence on the blood coagulation cascade and erythrocyte toxicity was observed. Cytotoxicity and genotoxicity safety were also found for these two novel sets of compounds.

Background Ischemic stroke is one of the leading causes of mortality and morbidity. The currently available non-invasive therapeutic options are not sufficiently efficacious. Post-ischemic brain is characterized by a prominent inflammatory response. Little is known about the involvement of cyclooxygenase (COX)-1 in the pathophysiology of ischemic stroke. Objective This study was undertaken to examine the effects of a highly selective COX-1 inhibitor – mofezolac – on clinical outcomes and brain inflammatory markers in post-stroke rats. Methods Stroke was induced by subjecting rats to permanent middle cerebral artery occlusion (MCAO). Control rats underwent a sham surgery. Rats were treated with mofezolac (50 mg/kg, intraperitoneally [ip]) once daily for 14 days. Control animals were treated with vehicle. Body temperature (BT), neurological score (NS) and cumulative mortality were monitored at different time points. At the end of the experiment, rats were euthanized and three brain regions (hypothalamus, hippocampus and frontal cortex) were extracted. Levels of interleukin (IL)-6, prostaglandin (PG)E2 and tumor necrosis factor (TNF)-α in these brain regions were determined by ELISA kits. Results BT, NS and cumulative mortality were all significantly higher in post-MCAO rats than in sham-operated rats, irrespective of the treatment given. BT, NS and mortality rate did not differ significantly between mofezolac-treated and vehicle-treated sham-operated animals. BT was significantly lower in mofezolac-treated as compared to vehicle-treated post-MCAO rats. Mofezolac did not significantly alter NS in post-MCAO rats at any time-point. Cumulative 14-day mortality was non-significantly higher in mofezolac-treated as compared to vehicle-treated post-MCAO rats (48 % vs. 21 %, respectively; P = 0.184). Mostly, IL-6 and TNF-α levels did not differ between post-MCAO and sham-operated rats and were not affected by mofezolac treatment. In contrast, mofezolac significantly decreased PGE2 levels in post-MCAO rats’ brains. Conclusion Overall, these results suggest that chronic treatment with the selective COX-1 inhibitor mofezolac did not reduce morbidity or mortality in post-stroke rats.

The diarylisoxazole molecular scaffold is found in several NSAIDs, especially those with high selectivity for COX-1. Here, we have determined the structural basis for COX-1 binding to two diarylisoxazoles: mofezolac, which is polar and ionizable, and 3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6) that has very low polarity. X-ray analysis of the crystal structures of COX-1 bound to mofezolac and 3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole allowed the identification of specific binding determinants within the enzyme active site, relevant to generate structure/activity relationships for diarylisoxazole NSAIDs.

The inhibition of gastric cyclooxygenase 1 (COX-1) enzyme was believed to be the major cause of non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastric ulcer. Recent studies disproved this belief and showed that the gastric tissues vulnerability is not solely connected to COX-1 inhibition. This work aimed at exploring and rationalizing the differential analgesic and anti-inflammatory activities of novel selective COX-1 inhibitors with improved gastric profile. Two novel series of 4,5-diarylthiazole and diarylimidazole were designed, synthesized in analogy to selective COX-1 inhibitors (mofezolac and FR122047) which lack gastric damaging effects. The new compounds were evaluated in vitro for their COXs inhibitory activity and in vivo for their anti-inflammatory and analgesic potentials. Four compounds; diphenylthiazole glycine derivatives (15a, 15b) and diphenylimidazolo acetic acid derivatives (19a, 19b), which possess carboxylic acid group exhibited significant activity and selectivity against COX-1 over COX-2. Of these compounds, (4,5-bis(4-methoxyphenyl)thiazol-2-yl)glycine 15b was the most potent compound against COX-1 with an inhibitory half maximal concentration (IC50) of 0.32μM and a selectivity index (COX-2 IC50/COX-1 IC50) of 28.84. Furthermore, an ulcerogenicity study was performed where the tested compounds demonstrated a significant gastric tolerance. Interestingly, the most selective COX-1 inhibitor showed higher analgesic activity in vivo as expected compared to their moderate anti-inflammatory activity. This study underscores the need for further design and development of novel analgesic agents with low tendency to cause gastric damage based on improving their COX-1 affinity and selectivity profile.

Biomarkers of specific targets are becoming an essential objective for clinical unmet clinical needs to improve diseases early detection and increase patient overall survival. Ovarian cancer is among the highest mortality gynecological cancers. It is asymptomatic and almost always diagnosed at advanced stage. At five years from the first diagnosis the survival rate of ovarian cancer patients is only 30%. Cyclooxygenase (COX)-1 as opposed to COX-2 is known to be overexpressed in ovarian cancer. Therefore, fluorescent probes targeting COX-1 were designed and prepared in fair to good yields for its quantitatively detection in human ovarian cancer cell lines (OVCAR-3 and SKOV-3). In particular, both cytofluorimetric and immunofluorescent experiments showed that N-[4-(9-dimethylimino-9H-benzo[a]phenoxazin-5-ylamino)butyl]-2-(3,4-bis(4-methoxyphenyl)isoxazol-5-yl)acetamide chloride (11) enters into OVCAR-3 cells and is mainly localized on the membrane containing the COX-1. Membrane fluorescence emission represents about 80% of the total fluorescence measured in the whole cell, while the non-specific labeling represents only 20%. This result indicates that the intensity of fluorescence emission is almost exclusively attributable to 11 bound to COX-1 located on the membrane. Furthermore, no diffusion inside the cell occurs. IC50 hCOX-1 value of 11 determined by measuring the O2 consumption during the bis-oxygenation of the arachidonic acid catalysed by COX-1 was found to be equal to 1.8 nM. Furthermore, 11 inhibits oCOX-1 with IC50 = 6.85 nM and mCOX-2 with IC50 = 269.5 nM; the corresponding selectivity index SI is equal to 39.3 against oCOX-1. 11 inhibits oCOX-1 at 0 min of incubation with 91% inhibition, whereas in the same time it does not inhibit mCOX-2. Fingerprints for Ligands and Proteins (FLAP) software calculations were performed to justify 11 higher COX-1 inhibitory potency than mofezolac (COX-1 IC50 = 5.1 nM), which in turn is a moiety of 11. Specifically, the two compounds bind differently in the COX-1 active site.

A set of novel diarylisoxazoles has been projected using mofezolac (1) as a lead compound to investigate structure-inhibitory activity relationships of new compounds and the cyclooxygenases (COXs) catalytic activity. Mofezolac was chosen because is the most potent and selective reversible COX-1 inhibitor [COX-1 IC50 = 0.0079 μM and COX-2 IC50 > 50 μM, with a selectivity index (SI) in favor of COX-1 higher than 6300]. Seventeen new compounds were synthesized in fair to good yields and evaluated for their COXs inhibitory activity and selectivity. SIs ranged between 1 and higher than 1190.3,4-Bis(4-methoxyphenyl)-5-vinylisoxazole (22) has the highest SI with COX-1 IC50 = 0.042 μM and COX-2 IC50 > 50 μM. 1 and 22 were superior to aspirin in inhibiting platelet aggregation (IC50 = 0.45, 0.63 and 1.11 μM, respectively) in human platelet rich plasma (hPRP) assay. They did not induce blood coagulation and hemolysis, and are neither genotoxic nor mutagen. 1 and 22 slightly increase bortezomib cytotoxic effect on multiple myeloma (MM) cell lines (NCI-H929 and RPMI-8226) and affects MM cell cycle and apoptosis when co-administered with the proteasome inhibitor bortezomib, a drug clinically used to treat plasma cell neoplasms including MM. In addition, structure-based binding mode of 1 and 22, through Fingerprints for Ligands and Proteins (FLAG) calculation, allowed to explain the one order of magnitude difference between COX-1 IC50 values of the two compounds. Specifically, the higher inhibitory potency seems due to the formation of a H-bond between COX-1 S530 and the carboxyl, present in 1 and absent in 22.

Few selective cyclooxygenase-1 (COX-1) inhibitors have been described up to now, although recent studies underlined the involvement of COX-1 in the carcinogenesis, pathogenesis of neuroinflammation, cardiovascular diseases and pain. Among the known COX-1 inhibitors none proved to be a good drug candidate, with the exception of mofezolac, that is clinically used as an analgesic drug. New selective inhibitors were very often discovered as a minor achievement during SAR investigations to discover selective COX-2 inhibitors (COXIBs). After a recognition of the new COX-1 inhibitors synthesized in the last five years, it was attempted to draw, for each chemical class, a structure which might highlight the determinant molecular features able to switch the selectivity towards the COX-1 isoform. Overall, this review could constitute a tool to a better design of novel selective COX-1 inhibitors, to be used in a disease theranostic approach targeting COX-1.

Neuroinflammation is the earliest stage of several neurological and neurodegenerative diseases. In the case of neurodegenerative disorders, it takes place about 15–20 years before the appearance of specific neurodegenerative clinical symptoms. Constitutive microglial COX-1 is one of the pro-inflammatory players of the neuroinflammation. Novel compounds 3, 14 and 15 (Galmof0, Galmof5 and Galmof11, respectively) were projected, and their synthetic methodologies developed, by linking by an ester bond, directly or through a C5 or C11 unit linker the highly selective COX-1 inhibitor mofezolac (COXs selectivity index > 6000) to galactose in order to obtain substances capable to cross blood-brain barrier (BBB) and control the CNS inflammatory response. 3, 14 and 15 (Galmofs) were prepared in good to fair yields. Galmof0 (3) was found to be a selective COX-1 inhibitor (COX-1 IC50 = 0.27 μM and COX-2 IC50 = 3.1 μM, selectivity index = 11.5), chemically and metabolically stable, and capable to cross Caco-2 cell monolayer, resembling BBB, probing that its transport is GLUT-1-mediated. Furthermore, Galmof0 (3) powerfully inhibits PGE2 release higher than mofezolac (1) in LPS-stimulated mouse BV2 microglial cell line, a worldwide recognized neuroinflammation model. In addition, Fingerprints for Ligands and Proteins (FLAP) was used to explain the different binding interactions of Galmofs with the COX-1 active site.

Activated microglia secrete an array of pro-inflammatory factors, such as prostaglandins, whose accumulation contributes to neuronal damages. Prostaglandin endoperoxide synthases or cyclooxygenases (COX-1 and COX-2), which play a critical role in the inflammation, are the pharmacological targets of non-steroidal anti-inflammatory drugs, used to treat pain and inflammation. Since it was reported that COX-1 is the major player in mediating the brain inflammatory response, the aim of this study was to evaluate the effects of highly selective COX-1 inhibitors, such as P6 and mofezolac, in neuroinflammation models. Lipopolysaccharide (LPS)-activated mouse BV-2 microglial cells and LPS intracerebroventricular-injected mice as in vitro and in vivo neuroinflammation models, respectively, were used to probe the antiinflammatory efficacy of P6 and mofezolac. Both P6 and mofezolac reduce COX-1 expression in LPS-activated BV-2 cells. This reduction was accompanied with PGE2 release reduction and NF-kB activation downregulation. Coextensively, in the in vivo model, both glial fibrillary acidic protein and ionized calcium-binding adapter molecule-1 expression, two markers of inflammation, were reduced by mofezolac to a rank depending on the encephalon area analyzed. The increase of COX-1 expression observed in all the brain sections of LPS-treated mice was selectively downregulated by the in vivo treatment with mofezolac as well as PGE2 release and Ikβα phosphorylation amount assayed in the brain areas tested. These results indicate the capability of P6 and mofezolac to modulate the NF-kB signaling pathway, emphasizing the neuroprotective effect and therapeutic potential of COX-1 inhibitors in the control of neuroinflammatory diseases.

We demonstrated recently that mofezolac, a cyclooxygenase-1 (COX-1) selective inhibitor, suppresses the development of azoxymethane (AOM)-induced colonic aberrant crypt foci in F344 rats and intestinal polyps in APC1309 mice. In the present study, we therefore investigated the effects of mofezolac on colon cancer development. Male F344 rats were injected subcutaneously with 15 mg/kg body weight of AOM in the back twice at 7-day intervals from 5 weeks of age, and fed a diet containing 600 or 1200 ppm mofezolac for 32 weeks, starting 1 day before the first dosing of AOM. Treatment with 1200 ppm mofezolac significantly reduced the incidence, multiplicity and volume of colon carcinomas to 79%, 2.15 ± 1.65 and 7.5 ± 11.8 mm3, respectively, compared with 94%, 3.19 ± 1.87 and 23.7 ± 31.2 mm3 in the AOM treatment alone. Administration of 600 ppm mofezolac showed only a slight reduction. No side effects were observed in any of the groups. These results confirm that COX-1, as well as COX-2, contributes to colon carcinogenesis and that mofezolac may be a good chemopreventive agent for human colon cancer. (Cancer Sci 2006; 97: 1011–1014)

Zaltoprofen, a preferential COX-2 inhibitor, exhibited a potent inhibitory action on the nociceptive responses induced by a retrograde infusion of bradykinin into the right common carotid artery in rats. However, other COX-2 preferential inhibitors such as meloxicam and etodolac did not exhibit any apparent action, and also, preferential COX-1 inhibitors mofezolac and indomethacin, COX-1 and COX-2 inhibitor loxoprofen sodium showed a weak effect. These non-steroidal anti-inflammatory drugs (NSAIDs) except for zaltoprofen, strongly inhibited an acetic acid-induced writhing response related to PGs based on COX-1, at lower doses. Zaltoprofen had a moderate inhibitory effect compared with those of the above-mentioned NSAIDs. These results suggest that the inhibitory effect of zaltoprofen on bradykinin-induced nociceptive responses is not explainable by the inhibition of cyclooxygenase (COX). So, we examined the inhibitory effect of zaltoprofen on bradykinin-induced nociceptive responses by performing several in vitro experiments. Zaltoprofen did not bind to B 1 and B 2 receptors in a radio-ligand binding assay. In the cultured dorsal root ganglion cells of mature mice, zaltoprofen completely inhibited the bradykinin-induced increase of [Ca 2+ ] i , which was inhibited by B 2 antagonist D-Arg-[Hyp 3 , Thi 5,8 , D-Phe 7 ]-bradykinin, but not by B 1 antagonist. In addition, the inhibition of zaltoprofen on the increase of [Ca 2+ ] i was observed even under extracellular Ca 2+ -free conditions. The above results suggest that zaltoprofen produces an analgesic action on bradykinin-induced nociceptive responses by blocking the B 2 receptor-mediated pathway in the primary sensory neurons. Taken together, these results suggest that zaltoprofen may serve as a potent and superior analgesic for the treatment of pain.

Postoperative peritoneal adhesion represents a major complication of surgery, but the molecular mechanism underlying pathogenesis of adhesion is not fully understood. The present study investigated the roles of cyclooxygenase (COX)-1 and COX-2 in peritoneal adhesion induced by scraping the surface of the cecum and abdominal wall in mice. Slight, but macroscopically observable, peritoneal adhesion was induced even on day 1, and the extent of adhesion reached a maximum on day 7 and beyond. COX-1 mRNA was constitutively expressed in the intact cecum, and its expression level was not altered after the mechanical stimulus. In contrast, expression of the COX-2 gene was markedly increased after the stimulus, and maximum expression was observed on days 3 to 7. Mofezolac, a specific COX-1 inhibitor, had no effect on peritoneal adhesion at 30 mg/kg and had only marginal effects on prostaglandin (PG)E2 levels in the cecum or peritoneal fluid. On the other hand, two highly selective inhibitors for COX-2, NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide) and CAY10404 [3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole], dose-dependently inhibited both adhesion formation and the increase in PGE2 levels (3-30 mg/kg). The effects of NS-398 were eliminated when PGE2 or (R)-butaprost was administered exogenously. A COX-2 antisense oligonucleotide also attenuated adhesion formation. Activation of p38 mitogen-activated protein (MAP) kinase was observed in the traumatized cecum, and an MAP kinase inhibitor, SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole], inhibited adhesion formation (54% inhibition at 15 microM) and also reduced the COX-2 mRNA level and PGE2 levels. In conclusion, COX-2, but not COX-1, plays a significant role in mechanical stimulus-induced peritoneal formation in the mouse cecum.

Objective and Design: Effects of cyclooxygenase inhibitors on noxious thermal stimuli were investigated in non-inflamed and inflamed rats.¶Materials: Male Sprague-Dawley rats were used in this study.¶Treatment: Cyclooxygenase inhibitors, indomethacin, mofezolac, NS-398, and JTE-522 were administered orally at a dose of 10 mg/kg 1 h prior to and 4 h after the intravenous injection of lipopolysaccharide (1 mg/kg).¶Methods: The nociceptive response was evaluated from the escape latency of foot withdrawal to the thermal stimuli with a beam of light. Expression of cyclooxygenase was examined by reverse transcription-polymerase chain reaction.¶Results: In normal rat, administration of indomethacin, relatively cyclooxygenase-1-selective inhibitor, mofezolac, or cyclooxygenase-2-selective inhibitors, NS-398 and JTE-522 had no effects on the escape latency against thermal stimuli. Injection of lipopolysaccharide into rat induced the expression of mRNA for cyclooxygenase-2 in the subcutaneous tissue of foot pad. The escape latency at 8 h was significantly shortened by the injection. This hyperalgesia could be reversed by pretreatment of rat with NS-398 or JTE-522, but not with mofezolac.¶Conclusions: Cyclooxygenases may have little participation in peripheral skin thermal nociception in non-inflamed condition, although cyclooxygenase-2 could be responsible for the hyperalgesia during inflammation induced by lipopolysaccharide.¶

Nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the production of primary prostanoids by blocking both cyclooxygenase (COX) -1 and COX-2 activities, and in doing so, not only decrease inflammation and pain, but also promote gastrointestinal tract damage and bleeding.The inhibitory activities of zaltoprofen (ZP) on the biosynthesis of prostaglandin E2 (PGE2) at the inflammatory site and stomach were compared with those of some NSAIDs (celecoxib : CL, meloxicam : ML, lornoxicam : LR, mofezolac : MF) using the rat carrageenin-air pouch model. At the dose of ZP and CL that inhibited PGE2biosynthesis at the inflammatory site, these hardly inhibited the PGE2release from stomach. In contrast, ML, MF and LR inhibited the biosynthesis of PGE2on both sites at the same dose.Furthermore these NSAIDs inhibited the carrageenin-induced paw edema, and produced the ulcer in a dose-dependent manner. The safety index, UD50/ED40ratio, of CL, ZP, ML, LR and MF was >103, 11.3, 0.8, 0.8 and 0.1, respectively. These safety indexes correlated well with the tissue selectivity of NSAIDs on PG biosynthesis inhibition.These results suggest that the PG biosynthesis at the inflammatory site and stomach of the same rat is a very useful parameter for evaluating NSAIDs. It seems that these properties of ZP were contributed to the low ulcerogenic activity on clinical uses.

Optimal dose of N-22 (Mofezolac), a new non-steroidal anti-inflammatory agent, was evaluated in a double-blind comparative study on post-exodontic pain, at 3 single dosing levels of 100 mg, 150 mg and 200 mg, respectively. The results are summarized as follows.1) A total of 197 cases subjected for analysis are broken down into a group of 68 cases with a 100mg dose, that of 64 eases with a 150mg dose and that of 65 cases with a 200mg dose, respectively. No significant deviation in clinical background factors of all cases was recognized.2) A total of 189 cases (67 cases-100mg, 60-150mg arid 62-200mg) were subjected for analysis of its efficacy. Analysis by U-test proved both 150 mg and 200mg groups to be significantly superior (P

Mofezolac (N-22) is a newly developed analgesic and anti-inflammatory agent. The acute toxicities of N-22 were investigated in ICR mice and Wistar rats in oral (p.o.), intraperitoneal (i.p.) and subcutaneous (s.c.) routes. LD50 values of N-22 in mice were 1528 mg/kg (p.o.), 275 mg/kg (i.p.) and 612 mg/kg (s.c.) for males, and 1740 mg/kg (p.o.), 321 mg/kg (i.p.) and 545 mg/kg (s.c.) for females. Those in rats were 920 mg/kg (p.o.), 378 mg/kg (i.p.) and 572 mg/kg (s.c.) for males, and 887 mg/kg (p.o.), 342 mg/kg (i.p.) and 510 mg/kg (s.c.) for females. The sex difference was not clearly observed in mice and rats, but the species difference was observed in p.o. routes. As an initial toxic sign, the hypoactivity was observed in mice and rats of all routes, subsequently, paleness of skin, anemic conjunctiva, emaciation, stupor and/or coma were observed in mice and rats of p.o. and i.p. routes. In rats of those routes, tonic and/or asphyxial convulsion and dyspnea were also observed. In pathological examination of mice and rats, gastrointestinal disorders were observed in p.o. and i.p. routes, and changes of subcutaneous tissue at the injection site were observed in s.c. route.

In vivo metabolism of[3, 4-di(4-methoxyphenyl)-5-isoxazolyl]acetic acid (mofezolac) was studied in mice, rats, dogs and monkeys following intravenous injection or oral administration, and serum protein binding of mofezolac and its metabolites were studied in rats, mice, dogs, monkeys and human.Obtained results were as follows;1. Following metabolites were identified in the rat urine and bile and dog bile ; [3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-5-isoxazolyl]acetic acid (3-DM-mofezolac), [4-(4-hydroxyphenyl)-3-(4-methoxyphenyl)-5-isoxazolyl]acetic acid (4-DM-mofezolac), [3, 4-di(hydroxyphenyl)-5-isoxazolyl]acetic acid (3, 4-DM-mofezolac), 4-[4-(4-methoxyphenyl)-5-carboxymethyl-3-isoxazolyl]phenyl hydrogen sulfate (3-DM-mofezolac-sulfate), 4-[(4-hydroxyphenyl)-5-carboxymethyl-3-isoxazolyl]phenyl hydrogen sulfate (3, 4-DM-mofezolac-sulfate), β-D-glucopyranuronic acid 1-[3, 4-di(4-methoxyphenyl)-5-isoxazolyl]acetate (mofezolac-glucuronide).2. After administration to various animal species, plasma levels of mofezolac reached Cmax rapidly except monkeys. Area under concentration time curve (AUC) of mofezolac shows the species related differences with the highest AUC value found in dogs. Main metabolite in mice and monkeys plasma were 3-DM-mofezolac and 3-DM-mofezolac and 3-DM-mofezolacsulfate was found only in rats. In dogs plasma only a small quantities of metabolites were observed in the plasma. AUC of 3-DM-mofezolac-sulfate in male rats was about three times higher than female rats.3. After oral administration of mofezolac to rats, the residue of mofezolac in the stomach was about 60% at 0. 5hr, 33% at 2hr. And at 8hr, the residue in the stomach was slightly detected. In the small intestine, its content consisted mainly of 3-DM-mofezolac-sulfate. In the large intestine, the content of 3-DM-mofezolac was increased when compared with that of the small intestine.4. After oral administration, main metabolites found in the urine were 3-DM-mofezolac in mice, 3-DM-mofezolac and 3-DM-mofezolac-sulfate in rats, 3-DM-mofezolac and unchanged drug in monkeys, and unchanged drug in dogs. Total amount of urinary excretion was the lowest in dogs among examined animal species.5. In the feces after oral administration, main metabolites were 3-DM-mofezolac in mice and rats, and unchanged drug in dogs.6. Mofezolac, 3-DM-mofezolac and 4-DM mofezolac were strongly bound to serum proteins of all species studied. Mofezolac was mainly bound to albumin of human serum.7. After administration to rats, total amount excreted with bile was about 39% in bile within 24 hours. Main metabolite was 3-DM-mofezolac-sulfate.

We studied the factors related to species and sex difference contributing to metabolic patterns of[3, 4-di(4-methoxyphenyl)-5-isoxazolyl] acetic acid (mofezolac) in vitro, and the metabolic pathway determined after administrations of metabolites of mofezolac. Also the metabolism of mofezolac in germ free rats, and the in situ absorption rates using recirculation perfusion of mofezolac and the metabolites were studied.1. Mofezolac demethylase activities in monkeys and rats were highest among investigated animals, following by the activity in mice. The activity in the dog was about 10 times lower than that in mice. Sex difference in mofezolac demethylase activity was not observed in the rats. UDP-glucuronatemofezolac glucuronosyltransferase activities in the monkey and the dog were high, and that in the rat was about 1/3 lower than that in the monkey and the dog.2. Mofezolac sulfotransferase activity in rats cytosol was much higher than the activities in other investigated animals. Sex deference was observed in rats ; the activity in male rats was about 3 times higher than that in female rats. Therefore, the total amount of sulfoconjugate of[3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-5-isoxazolyl] acetic acid(3-DM-mofezolac) in vivo reflects the sulfotransferase activity of liver.3. After administrations of mofezolac metabolites, the main metabolite in bile derived from 3-DM-mofezolac was 4-[4-(4-methoxyphenyl)-5-carboxymethyl-3-isoxazolyl] phenyl hydrogeo sulfate (3-DM-mofezolac-sulfate), and the main metabolite derived from[4-(4-hydroxyphenyl)-3-(4-methoxyphenyl)-5-isoxazolyl]acetic acid (4-DM-mofezolac) was[3, 4-di(hydroxyphenyl)-5-isoxazolyl] acetic acid (3, 4-DM -mofezolac-sulfate). These results explain that 3-(4-methoxyphenyl) was desmethylated easier than 4-(4-methoxyphenyl). No sulfoconjugate of 4-DM-mofezolac was formed after administration of 4-DM-mofezolac. This result suggested that sulfoconjugate was not formed by binding of a sulfate directly to OH in the phenyl group on the side of 4-position of isoxazol.4. The main fecal metabolite in SPF rats was 3-DM-mofezolac, while in germ free rats was 3-DM-mofezolac-sulfate. This result clarified that 3-DM-mofezolac-sulfate was degraded by intestinal microflora.5. The absorption rate constant of mofezolac from the small intestine was close to the absorption rate constants of indomethacin or salicylic acid using in situ recirculation perfusion system. In the stomach, mofezolac was absorbed in about 1/2 of that in small intestine when the perfusate was acidic (pH3). The absorption rate constant of 3-DMmofezolac-sulfate was about 8 times lower than the that of mofezolac. 3-DM-mofezolacsulfate was hardly absorbed.

A micronucleus test using BDF1 male mice was conducted in order to evaluate the in vivo mutagenicity of mofezolac (N-22). N-22 was orally administered at doses of 75, 150, 300 and 600 mg/kg, with a sampling time of 24 hr. The frequency of polychromatic erythrocytes with micronuclei was 0.07-0.08% in the groups treated with N-22 and did not differ markedly from that of the vehicle control (0.13%). The present study indicates that N-22 has no in vivo mutagenic properties.

The absorption, distribution and excretion of [3, 4-di(4-methoxyphenyl)-5-isoxazolyl] acetic acid (mofezolac) were studied in rats after a single or repeated oral administration of 14C-labeled-mofezolac.1. Concentration of radioactivity in the blood increased rapidly after oral administration, reaching Cmax within 15min, and decreased to 1.4% of the maximum level 12hr after administration.2. Approximately 55% and 39% of radioactivity were excreted during 72hr after administration in urine and feces, respectively.3. About 62% of radioactivity was excreted in bile. When the bile was administered, about 52% of radioactivity present in the bile was re-absorbed. This result suggested the presence of the entero-hepatic circulation.4. Tissue radioactivity was higher in the stomach, liver and kidney, while the radioactivity in other tissues was lower than that in plasma. Radioactivity disappeared rapidly from any tissue.5. When female rats were administered 14C-mofezolac on day 11 or 18 of pregnancy, or slight radioactivity usually lower than in maternal plasma was observed in the fetus. Radioactivity in fetus as well as in maternal tissues disappeared rapidly. Radioactivity in the fetus was not detected at 24hr after administration.6. Concentration of radioactivity in the milk at 2hr after administration to lactating rats reached levels about 5 times higher than that in plasma, however the radioactivity in milk was not detected at 48hr.7. In case of repeated administration of 14C-mofezolac for 7 days, the disposition of radioactivity was almost same as the result after a single dosing, and radioactivity scarcely remained in any tissues after the last administration.

Mofezolac (N-22) is a new developed analgesic and anti-inflammatory agent. A teratogenicity study of N-22 was carried out in New Zealand White rabbits to examine the effect on the dams and the teratogenic potentiality. N-22 was administered orally at the dose levels of 12.5, 50 and 200 mg/kg during the organogenesis from day 6 to day 18 of gestation. All pregnant females were sacrificed on day 28 of gestation and their fetuses were examined. There were no growth retardation and teratogenic effects on fetuses from the dams administered N-22, although the administration of 200 mg/kg produced a decrease in food consumption of the dams concomitant with the embryocidal effects as shown by an increase in the early resorption rate. Thus, non-toxic dose level of N-22 on the dams and fetuses was considered to be 50 mg/kg.

Antigenicity studies of mofezolac (N-22) were examined in mice and guinea pigs and the following results were obtained. The findings of active systemic anaphylaxis, passive hemagglutination test, 4 hour passive cutaneous anaphylaxis (4-hr PCA) and 8-day PCA in guinea pigs revealed that N-22 possessed neither immunogenic nor eliciting potentiality. However, N-22 was shown to be eliciting antigenicity in mice when given N-22-ovalbumin conjugate plus Freund's complete adjuvant (FCA) as immunogen.

A three-month oral subacute toxicity study of mofezolac (N-22), a non-steroidal anti-inflammatory agent, was performed using dose levels of 6, 20, 60 and 200 mg/kg in rats, and recovery was also assessed one month after withdrawal. 1. Toxic signs caused by N-22 administration, observed only in the 200 mg/kg group, were as follows: soiling around the mouth and/or nose, piloerection, anemia, diarrhea, emaciation and decreased spontaneous locomotor activity. Nine males and thirteen females in the 200 mg/kg group excreted bloody diarrhea and died of general exhaustion between weeks four and thirteen of study. 2. In the 200 mg/kg group, decrease in food consumption and suppression of body weight gain were noted in males from about week four and in females from about week six after initiation of administration, and increase in water consumption was noted in males from about week seven. 3. Urinary examination revealed a decline in urinary pH in males of the 20 mg/kg and above groups and elevation of urobilinogen levels in males of the 60 and 200 mg/kg groups. 4. Hematological examination showed decreases in erythrocyte count (RBC), hematocrit value (Ht) and hemoglobin concentration (Hb) and increase in reticulocyte rate in both sexes of the 200 mg/kg group and an increase in neutrophil rate in males of the 200 mg/kg group. 5. Biochemical examination demonstrated a decrease in chloride (Cl-) in males receiving the 20 mg/kg or above doses and a decrease in calcium (Ca++) in males of the 60 and 200 mg/kg groups. Moreover, there were decreases in cholinesterase (ChE) activity, total protein (TP) and albumin (Alb) values, as well as increases in blood urea nitrogen (BUN), uric acid (UA) and potassium (K+) in both sexes of the 200 mg/kg group, along with elevations in GOT and lactate dehydrogenase (LDH) activities in females of the 200 mg/kg group. 6. The absolute and/or relative organ weights for liver, kidneys, spleen and adrenals were increased in the 200 mg/kg group. 7. On pathological examination, perforating ulceration in the jejunum and ileum, turbid ascites, adhesion and inflammatory changes in capsules of the abdominal organs, splenomegaly, mesenteric lymph node hyperplasia and inflammatory changes in the thoracic cavity were observed in dead animals of the 200 mg/kg group. Similar pathological changes were observed in a few survival cases of the 200 mg/kg group. 8. After a one month recovery period, the above-mentioned changes had mostly recovered, indicating that they were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical usefulness of N-22 (Mofezolac; N), a new non-steroidal antiinflammatory agent, on post-exodontic pain was evaluated at a single dose level of 150mg/time, in a double-blind comparative study, in comparison with Mefenamic acid; M also at a single dose level of 500 mg/time; results are summarized as follows.Of a total of 228 cases, 33 with no onset of post-exodontic pain took neither of the agents and were therefore removed, meaning that 195 cases were subjected for analysis, in comparison with M put under previously well controlled studies. No significant difference in patient background factors between N and M groups was found. Based on the protocol, either N or M was administered alone for evaluation at a single dose level/time. Double dummy method was used in allotment for administration of 4 kinds of test agents-N, NI, N's placebo and M's placebo.Of a total of 193 efficacy evaluable cases (N-96 & M-97), 25.0% of N group and 11.3% of M were rated “Remarkably improved” in final global improvement rate, with N group found significantly superior (P

Metabolism of [3, 4-di(4-methoxyphenyl)-5-isoxazolyl]acetic acid(mofezolac) was studied in rats after oral administration of 14C-labeled mofezolac.1. After administration of 14C-mofezolac, more than 90% of the radioactivity present in plasma accounted for mofezolac and identified metabolites, [3-(4-hyd roxyphenyl)-4-(4-methoxyphenyl)-5-isoxazolyl] acetic acid (3-DM-mofezolac), [4-(4-hydroxyphenyl)-3-(4-methoxyphenyl)-5-isoxazolyl]acetic acid (4-DM-mofezolac), [3, 4-di(hydroxy-phenyl)-5-isoxazolyl]acetic acid (3, 4-DMmofezolac), 4-[4-(4-methoxyphenyl)-5-carboxymethyl-3-isoxazoly]phenyl hydrogen sulfate (3-DM-mofezolac-sulfate), 4-[4-(4-hydroxyphenyl)-5-carboxymethyl-3-isoxazolylpheny] hydrogen sulfate(3, 4-DM-mofezolac-sulfate). 2. After administration of 14C-mofezolac, more than 80% of the radioactivity present in urine (0-24hr) or bile (0-6hr) accounted for mofezolac and identified metabolites (3-DM-mofezolac, 4-DM-mofezolac, 3, 4-DM-mofezolac, 3-DM-mofezolac-sulfate and 3, 4-DM-mofezolac-sulfate). The largest quantity of unknown metabolite was only 1.4% of dose. These results suggested that the disposition of mofezolac in rats could be account for identified metabolites.3. In feces, the radioactivity of identified metabolites contributed to about 50% of total radioactivity.

As with cyclooxygenase (COX)-2, genetic disruption of COX-1 gene or pharmacologic inhibition of its activity has been shown to decrease the number of intestinal polyps in Apc gene-deficient mice. The present study was designed to investigate the combined effects of COX-1 and COX-2 selective inhibitors on spontaneous polyp formation in APC1309 female mice. The animals were treated with 300 or 600 ppm mofezolac (a COX-1 selective inhibitor) alone, 200 or 400 ppm nimesulide (a COX-2 selective inhibitor) alone, 300 ppm mofezolac plus 200 ppm nimesulide, 600 ppm mofezolac plus 400 ppm nimesulide, or 10 ppm indomethacin (a dual-COX inhibitor) in the diet from 7 weeks of age for 4 weeks. Percentage inhibition of polyp area in the intestine was 17% with 600 ppm mofezolac alone and 25% with 400 ppm nimesulide alone, their sum of 42% being almost equal to the 37% observed for the combination treatment. Administration of 300 ppm mofezolac plus 200 ppm nimesulide also significantly decreased polyp area in the intestine by 30%. Moreover, the numbers of polyps more than 2.5 mm in diameter were markedly decreased by combined treatment of both COX inhibitors. With 10 ppm indomethacin, the dual inhibitor, polyp area was also clearly reduced by 46%. Our results indicate that COX-1 and COX-2 may to some extent contribute to polyp formation independently and inhibitor combination treatment thus has particular potential for chemoprevention of colon carcinogenesis. © 2004 Wiley-Liss, Inc.

Involvements of kinin and prostaglandin and their interaction in noxious thermal stimuli were investigated in noninflamed and inflamed rats. The nociceptive response was evaluated from the escape latency of foot withdrawal to the thermal stimuli with a beam of light. The escape latency in kininogens-deficient Brown Norway (B/N-) Katholiek rats was significantly longer than that in the normal strain, B/N-Kitasato rats. The latency in B/N-Kitasato rat was prolonged by administration of a bradykinin (BK) B2 receptor antagonist, FR173657 (30 mg/kg, p.o.), whereas it was shortened by pretreatment with a kininase II inhibitor, captopril (10 mg/kg, i.p.). Both agents did not affect the latency in B/N-Katholiek rats. In normal Sprague-Dawley (SD) rat, administration of indomethacin did not change the escape latency against the thermal stimuli. In contrast, administration of indomethacin or a relatively cyclooxygenase-1-selective inhibitor, mofezolac (10 mg/kg, p.o.) significantly reduced numbers of writhing reaction in mice induced by acetic acid solution. Injection of lipopolysaccharide (1 mg/kg, i.v.) resulted in shortening escape latency at 8 h after the injection in B/N-Kitasato rats. This hyperalgesia could be reversed by pretreatment of the rats with indomethacin, a cyclooxygenase-2-selective inhibitor JTE-522 (10 mg/kg, p.o.), or FR173657, but not with mofezolac. The hyperalgesia was not seen in B/N-Katholiek rats. These results indicate that kinin has major participation in peripheral skin thermal nociception under noninflamed condition, although cyclooxygenases may have little participation. Prostaglandins produced by cyclooxygenase-2 could coordinate with BK to elicit hyperalgesia during inflammation induced by lipopolysaccharide.

COX-2 is an inducible cyclooxygenase (COX) that has been reported to be expressed in the macula densa and surrounding cortical thick ascending limb in normotensive rats. The present study assessed the contribution of COX-2 in three different rat models of hypertension, each characterized by a different activation of the renal renin-angiotensin system. Mean blood pressure (MBP) in the rat 2 kidney-1 clip (2K1C) model was significantly reduced with a COX-2 selective inhibitor, NS-398 (10 mg/kg, p.o., twice a day) (vehicle-administered rats (n = 8): 154 +/- 6 mmHg; NS-398-administered rats (n = 5): 128 +/- 10 mmHg). By contrast, a COX-1 selective inhibitor, mofezolac, did not lower MBP. Increased plasma renin activity (23 +/- 8 ng/kg/h (n = 6) vs. sham operation, 2.4 +/- 0.9 ng/kg/h (n = 4)) was markedly reduced to 6.8 +/- 2.7 ng/ml/h (n = 5) by NS-398, but not by mofezolac. The development of 1 kidney-1 clip (1K1C) hypertension was also inhibited by NS-398 (vehicle (n = 12): 133 +/- 1 mmHg; NS-398 (n = 7): 122 +/- 3 mmHg) accompanied by a reduction in plasma renin activity (3.0 +/- 0.3 ng/ml/h, n = 4) to 1.0 +/- 0.2 ng/ml/h (n = 5). The COX-2 inhibitor increased urinary excretions in the 1K1C model, but not in the 2K1C model. In a deoxycorticosterone acetate (DOCA)-salt model, plasma renin activity was markedly suppressed to less than 0.3 ng/ml/h. The COX-2 inhibitor caused no significant changes in MBP, plasma renin activity, or urinary excretion, suggesting that COX-2 made a lesser contribution in this model. Increased expression of COX-2 mRNA and protein was observed in the kidneys of 1K1C and 2K1C rats, but not in DOCA-salt rats. These results suggest that COX-2 plays a significant role in the development of 2K1C and 1K1C renovascular hypertension, in addition to making a substantial contribution to the diuretic effect in the 1K1C model.

Cyclooxygenase (COX)-2, one enzyme isoform responsible for producing prostanoids from arachidonic acid, contributes to colon carcinogenesis. Recently, genetic disruption of COX-1, the other isoform, was shown to decrease the number of intestinal polyps and prostaglandin E(2) levels in intestinal mucosa, like the case with COX-2 gene disruption, in Min mice. We therefore investigated whether a COX-1 selective inhibitor, mofezolac, suppresses intestinal carcinogenesis in rodents. F344 male rats, receiving azoxymethane (AOM, 15 mg/kg body wt) s.c. injections at 5 and 6 weeks of age, were fed a diet containing 600 or 1200 p.p.m. mofezolac for 4 weeks. The number of aberrant crypt foci (ACFs) per rat and the bromodeoxyuridine labeling index of the crypt epithelium were dose-dependently decreased by administration of mofezolac, the value for the former at 1200 p.p.m. being 60% of control value. When Apc gene knockout mice (APC1309 mice) were given 600 or 1200 p.p.m. mofezolac in their diet for 8 weeks, the numbers of intestinal polyps were also dose-dependently decreased, with reduction to 59% of that in the control diet group at the higher dose. Nimesulide, a COX-2 selective inhibitor used as positive control, showed similar suppressive effects on the development of ACFs in AOM-treated rats and polyps in Apc gene knockout mice. The data indicate that both COX-1 and COX-2 can contribute to intestinal tumorigenesis.

Intraperitoneal injection of phenyl- p -benzoquinone (phenylquinone, PQ) induced writhing in mice for up to 30 min. During this time, the peritoneal content of 6-keto-prostaglandin F 1α (6-keto-PGF 1α ), a stable degradation product of PGI2, was highest in the 15-min. sample. In the peritoneal cells, the mRNA expression for the constitutive cyclooxygenase-1 (COX-1) was unchanged by PQ administration. In contrast, little mRNA for COX-2 was detected in the peritoneal cells from unstimulated animals, and was induced 60–120 min. after PQ administration. PGs involved in the induction of writhing thus seem to be derived from a COX-1 reaction. Oral administration of mofezolac, a non-steroidal anti-inflammatory drug which potently inhibits COX-1, suppressed the PQ-induced writhing and peritoneal accumulation of PGs without affecting mRNA expression for both COX isoforms in mice.

The effects of nonsteroidal anti-inflammatory drugs (NSAIDs), mofezolac, indomethacin, sodium diclofenac, and zaltoprofen, on the production of interleukin-1 receptor antagonist (IL-1ra) were examined in cultured human peripheral blood mononuclear cells (PBMC). Among the NSAIDs tested, mofezolac and sodium diclofenac were found to stimulate the mRNA expression for IL-1ra without affecting the mRNA expression for IL-1 beta. These two drugs also stimulated the secretion of IL-1ra by PBMC in the absence of bacterial lipopolysaccharide (LPS), however, the stimulatory effect of sodium diclofenac diminished in the presence of LPS. Mofezolac suppressed the mRNA expression for IL-1 beta in PBMC stimulated with exogenous IL-1 beta, indicating the secreted IL-1ra in the presence of mofezolac to be biologically active. Since IL-1ra suppresses the function of IL-1, a pro-inflammatory cytokine, the stimulatory effect of such NSAIDs as mofezolac on IL-1ra production could also be one of the mechanisms involved in its anti-inflammatory and antinociceptive actions.

We investigated the relationship between c-fos expression in the hind brain and peritoneal prostaglandin (PG) synthesis after visceronociceptive stimulation with acetic acid in rats. Time-course studies on the mRNA expression for c-fos in the hind brain and cyclooxygenase (COX) isoforms in the peritoneal cells, as well as on the peritoneal 6-keto-PGF1alpha accumulation, after stimulation indicated that COX-1 but not COX-2 was responsible for the peritoneal synthesis of PGs which were suggested to evoke c-fos expression in the hind brain. Pharmacological experiments using mofezolac, a preferential inhibitor against COX-1, and NS-398, a selective inhibitor against COX-2, confirmed the involvement of COX-1 derived PGs in the induction of c-fos expression in the hind brain following the noxious stimulation.

Mofezolac (N-22), a newly developed analgesic and anti-inflammatory agent, at dose levels of 0, 10, 30 and 90 mg/kg/day were administered orally to males from pre-mating to mating period and to females from pre-mating to early gestation period. Effects on reproductive performance of both sexes, especially reproductive capability, and development of offspring were examined. 1. In male parents, no changes in body weight and food consumption were found in all male groups, but the increase in gastric mucosal lesions such as ulcers were observed in 30 and 90 mg/kg/day groups. 2. In female parents, the decrease in body weight and food consumption of 90 mg/kg/day group during early gestation period were found, but at necropsy no changes were shown in all female groups. 3. Reproductive capability, mating and pregnancy performance were not affected. 4. No effects of N-22 on fetuses were observed. 5. The suggestions were as follows: No effect dose levels (NOELs) for male and female general-toxicologically were 10 and 30 mg/kg/day, respectively. NOELs for reproductive capability and for fetal development were 90 mg/kg/day.

1. The reverse mutation test was carried out on mofezolac (N-22) at dose range of 50-5000 micrograms/plate using Salmonella typhimurium strains, TA100, TA98, TA1535 and TA1537, and Escherichia coli strain WP2uvrA. In all tester strains no significant increases were observed in the number of revertant colonies as compared with solvent control in the absence or presence of mammalian metabolic activation system. 2. The chromosomal aberration test on N-22 was carried out using cultured Chinese hamster lung cells (CHL). The cells were treated with N-22 at the doses of 37.5, 75.0, 150 and 300 micrograms/ml without S9 Mix and at the doses of 150, 300, 450 and 600 micrograms/ml with S9 Mix. No significant differences were found in the incidence of structural-and numeral-aberrations of chromosomes as compared with the solvent control in the system without S9 Mix. However, in the system with S9 Mix, structural aberration (11.5%) and numeral aberration (14.2%) of chromosomes were observed in the groups of dosing 600 micrograms/ml with dose dependency. 3. These results indicate that N-22 has clastogenic activity by the metabolic activation.

The chronic toxicity study of mofezolac (N-22), a new developed analgesic and anti-inflammatory agent, was carried out in beagle dogs by oral administration of the test compound at dose levels of 1, 3, 6 and 12 mg/kg once daily for 52 weeks. The recovery study was also carried out by withdrawal for 5 weeks. 1. No death occurred during the administration and recovery periods in all groups, there were no abnormal findings due to N-22 administration on behavior and appearance, body weight and food consumption. 2. There were no abnormal findings due to N-22 administration on fecal test, urinalyses, hematological and biochemical examinations, hepatic and renal function tests, ophthalmological examination, electrocardiography, blood pressure and organ weight. 3. By histopathological examination, loss of villi in small intestine was found in one female of 6 mg/kg group, erosion in small intestine in one male of 12 mg/kg group, and shortening of villi and ulcer in small intestine in each one female of 12 mg/kg 4. Accordingly, the non-effective dose levels of N-22 were estimated to be 6 mg/kg for male and 3 mg/kg for female in this study.

Pharmacological activities of mofezolac were investigated in experimental animal models and compared with those of indomethacin, ibuprofen, mefenamic acid, aspirin and aminopyrine. Mofezolac showed a potent suppression of various writhing models in mice or rats; and its potency was slightly lower than that of indomethacin, but was higher than those of the other reference drugs. Thus, mofezolac was especially active against chemically induced writhing and also in the phenylquinone induced intraperitoneal dye leakage reaction in mice. Mofezolac also has a potent inhibitory activity on the algesic responses induced by the mechanical stimulus of the inflammed tissue. Mofezolac exhibited a therapeutic effect comparable to indomethacin in the urate synovitis in dogs. Considering the anti-inflammatory and antipyretic actions, mofezolac was obviously less effective than indomethacin, and its potency was similar to that of ibuprofen. The ulcerogenic effect of mofezolac on the gastric mucosa was far weaker than that of indomethacin. In in vitro studies, the prostaglandin biosynthesis and platelet aggregation were inhibited to the same extent by both mofezolac and indomethacin. Accordingly, it may be considered that the actions of mofezolac are due to the inhibition of cyclooxygenase. Our result suggest that mofezolac can be a useful drug that shows a rapid pain-relieving activity in acute inflammations.