Antinociceptive Activity of a Novel Non-Steroidal Anti-Inflammatory Drug (M-501 1) with Low Ulcerogenic Effects in Mice

Naofumi Murakami, Hiroshi Takase, Takako Tomita, Kazumi Iwata and Tomohiro Naruse

Research and Development Laboratories, Maruho Co., Ltd., 1-8-23 Oyodo Naka, Kita-ku, Osaka 531, Japan

Received February 16, 1996 Accepted June 14, 1996

ABSTRACT-Both analgesic and ulcerogenic activities of d-2-[4-(3-methyl-2-thienyl)phenyl] propionic acid (M-5011), a novel non-steroidal anti-inflammatory drug (NSAID), were compared with those of indometh- acin (IND), ketoprofen (KP), diclofenac sodium (DIF), zaltoprofen (ZLT) and tiaprofenic acid (TIA) in mice. All orally administered NSAIDs including M-5011 inhibited kaolin-induced writhing in a dose- dependent manner. M-5011 had an effective antinociceptive activity (ED50 value) of 0.63 mg/kg, being more potent than ZLT (16.80 mg/kg) and TIA (4.78 mg/kg), equipotent to DIF (0.68 mg/kg), and less potent than IND (0.21 mg/kg) and KP (0.28 mg/kg). All drugs tested significantly reduced peritoneal 6-keto- prostaglandin Fla (6-keto-PGFIa) levels at the peak kaolin-induced writhing time (7.5 min post-kaolin in- jection) without affecting peritoneal bradykinin (BK) levels. Antinociceptive effects of all drugs were closely correlated with inhibition of peritoneal 6-keto-PGFIa levels. Ulcerogenic activities (UD50 value) of M-5011 in the stomach and small intestines were 88.23 and 46.09 mg/kg, respectively. UD50 values of other drugs in the stomach and small intestines were as follows: 8.96 and 4.78 mg/kg, 20.04 and 10.75 mg/kg, 4.19 and 2.24 mg/kg, 62.86 and 46.55 mg/kg, and 110.92 and 54.78 mg/kg for IND, KP, DIF, TIA, and ZLT, respectively. Thus, the safety indexes (UD50/ED50) of the stomach (or small intestine) for M-5011, IND, KP, DIF, TIA and ZLT were 140.05 (73.16), 42.67 (22.76), 71.57 (38.39), 6.16 (3.29), 13.15 (9.74) and 6.60 (3.26), respectively. These findings suggest that M-5011 is a useful NSAID that shows potent antinociceptive effects with low ulcerogenic activities.

Keywords: M-5011, Antinociceptive activity, 6-keto-prostaglandin Fla, Bradykinin, Ulcerogenic activity

Acidic nonsteroidal anti-inflammatory drugs (NSAIDs) intestinal ulcerogenesis, which is derived from inhibition such as aspirin, indomethacin (IND), ketoprofen (KP) of PGs biosynthesis (6). To date, incessant efforts have and diclofenac sodium (DIF), which show potent anti- been directed towards the development of potent NSAIDs inflammatory effects, are extensively used for the therapy devoid of ulcerogenicity. Kaolin-induced writhing, main- of severe inflammatory diseases such as rheumathrosis ly mediated by both endogenous bradykinin (BK) and (1). The underlying mechanism of anti-inflammatory PGI2, is one of the reliable and simple tests for the evalu- effects of acidic NSAIDs is mainly attributed to inhibition ation of analgesic agents (7). The present study was un- of the excessive production of prostaglandins (PG) at dertaken to clarify both the analgesic effects on this inflammatory sites (2, 3). Recently, we have innovated a chemonociceptive response and gastrointestinal ulcero- novel acidic NSAID, d-2-[4-(3-methyl-2-thienyl)phenyl] genicity of M-5011 in mice compared with those of other propionic acid (M-5011). In the preliminary study, M- acidic NSAIDs. 5011 elicited potent anti-inflammatory effects in ultra- violet-induced erythema and carrageenin-induced edema tests, and these effects are at least equipotent to those of IND. Animals Apart from the anti-inflammatory effects, acidic Both male ICR (3 weeks; Shizuoka Laboratory Center, NSAIDs possess analgesic activities (4, 5). However, the Hamamatsu) and ddY mice (4 weeks, Shizuoka Labora- use of acidic NSAIDs for the relief of pain accompanied tory Center) housed in individual cages with free access to by inflammation is limited by their undesirable gastro- food and water were kept in a room maintained at con- min (0-15 min post-injection of kaolin). Complete and incomplete extension of either hind limb was regarded as one and one-half writhing responses, respectively. The inhibition (%) was calculated as follows:

Inhibition (%) =(A - B)/A x 100

Fig. 1. Structure of M-5011, d-2-[4-(3-methyl-2-thienyl)phenyl] where A = number of writhing responses in the controls propionic acid. *: Asymmetrical carbonate. and B =number of writhing responses in drug-treated animals. stant temperature (23±2C) and relative humidity Assay of bradykinin and 6-keto-PGFIa (55:±1007o) with an alternating 12-hr light/dark cycle Mice were sacrificed by exsanguination at various times (lighting on at 07:00 a.m.) after kaolin injection, and the peritoneal cavity was surgically exposed to collect the exudate. To inhibit the Drugs breakdown and production of kinins during exudate col- The chemical structure of M-5011 (Maruho Co., Ltd., lection (9), 2 ml of saline-ethanol (80 : 1) containing 62.5 Osaka) is shown in Fig. 1. Indomethacin (IND), pg of soybean trypsin inhibitor (Sigma Chem. Co.), 62.5 ketoprofen (KP) and diclofenac sodium (DIF) (Sigma pg of aprotinin (Sigma Chem. Co.), 17.5 pg of polybrene Chem. Co., St. Louis, MO, USA) were used as reference (Sigma Chem. Co.), 75.0 pg of disodium ethylenediamine drugs. Zaltoprofen (ZLT) and tiaprofenic acid (TIA) tetraacetate (Dojin Chemical Co., Kumamoto) and 42.5 were respectively extracted from Soleton® (Nippon pg ortho-fenanthroline (Nacalai Tesque, Kyoto) were Chemiphar Co., Ltd., Tokyo) and Surgam® (Nippon injected into the peritoneal cavity immediately after ex- Roussel, Tokyo) at our laboratories. Briefly, a mixture of sanguination. The peritoneal fluids collected with a poly- each drug with ethyl acetate was refluxed and then ethylene pipet was placed in a polypropylene tube before filtered. The filtrate was evaporated to dryness with a adding ethanol (final concentration: 80%). After standing rotating evaporator. The residue was recrystallized from for 1 hr at 0'C, the mixture was centrifuged at 1,500 x g toluene. The purities of ZLT and TIA determined by for 15 min at 4C. The isolated supernatant was evapo- high-performance liquid chromatography were 99.7% rated to dryness at 37 C with a rotating evaporator. and 99.9%, respectively. Desiccated samples were kept at - 801C until assay. Desiccated samples dissolved in 500 p1 of distilled water Preparation of test drugs were treated with 1/5 vol. of 20% trichloroacetic acid All drugs for dosing were freshly prepared at 7-day (TCA) to remove protein prior to centrifugation at intervals. These drugs were suspended in 0.5% carboxy- 1,500 x g for 10 min at 4C. The supernatants were col- methylcellulose sodium (CMC) (Wako Pure Chemical lected and diluted at 1 : 1 with an appropriate buffer Co., Osaka) at the highest concentration possible. Lower (buffer B of the MARKIT-M-Bradykinin®; Dainippon concentrations were then prepared by serial dilution Pharmaceutical, Osaka) for enzyme immunoassay with whenever and wherever applicable. an assay kit (MARKIT-M-Bradykinin®), which is based on a competitive reaction between the enzyme-labeled Kaolin-induced writhing response bradykinin (BK) and kinins in the BK antibody. The kinin The chemonociceptive response was performed as concentrations of samples were expressed in terms of the previously described with a slight modification (8). Male standard BK concentration. The lower and upper detec- ICR mice (4 weeks) were fasted overnight (fasted weight tion limits of BK were within 0.1-100 ng/ml, and the under 22 g) with free access to water before the experi- recovery rate of BK throughout the collection of ments. Groups of 10- 14 mice each were administered peritoneal washing was 60.5-L0.63% (mean ± S. E. , with M-5011 (3.00, 1.00, 0.30, 0.10 or 0.03 mg/kg), IND n=3). Inhibition (%) was calculated as follows: (1.00, 0.30, 0.10 or 0.03 mg/kg), KP (1.00, 0.30, 0.10 or Inhibition (%) =(A - B)/A x 100 0.03 mg/kg), DIF (3.00, 1.00, 0.30, 0.10 or 0.03 mg/kg), TIA (10.0, 3.0, 1.0, 0.3 or 0.1 mg/kg), ZLT (30.0, 10.0, where A=BK levels in the control group of each test and 3.0 or 1.0 mg/kg) or the vehicle (CMC) orally. One hour B = BK levels in the drug-treated group. later, kaolin (Wako Pure Chemical Co.) suspension at The 6-keto-PGFIa contents were determined by exu- 2.5 mg/0.5 ml in saline containing 0.05 mg of captopril dates of another kaolin-injected group. Saline solution (2 (Sigma Chem. Co.) was injected i.p. to induce writhing. ml) containing 1.4 x 10-4 M indomethacin (Sigma Chem. The chemonociceptive responses were measured for 15 Co.) was injected i.p. immediately after exsanguination to inhibit formation of PGs in the peritoneal cavity dur- Statistical analyses ing exudate collection. At designated intervals after the Results are expressed as the mean±S.E. Statistical initiation of kaolin-induced writhing, exudates were com- significance between the differences of means was verified pletely collected in polypropylene tubes. The samples by the multiple comparison test (Williams method). ED50 were centrifuged at 1,500 x g for 10 min at 41C before the values for kaolin-induced writhing (doses required to supernatants were frozen immediately under a stream of inhibit 50% of the response) and ID50 values (doses re- nitrogen gas and stored at - 80V until assay. Extraction quired to inhibit 50% of 6-keto-PGFIa by kaolin produc- of 6-keto-PGFIa was performed by the Powell method tion) were calculated by linear regression analysis using (10) with a slight modification. The samples were acidified inhibition percentages. UD50 values and 95% confidence to pH 3.0 with 1 N HCl and loaded onto a SEP-PAK C18 limits (CL) of ulcerogenicity were determined by probit cartridge (Waters Associates, Milford, MA, USA) pre- analysis using the ulceration incidence. viously washed with 5 ml each of methanol and distilled water. The cartridges were then washed serially with 5 ml each of distilled water, ethanol and benzene, before 6- keto-PGFIa was finally eluted with 5 ml of ethyl acetate - Effects of NSAIDs on kaolin-induced writhing methanol (9 : 1). The eluant were evaporated to dryness Total writhing responses for 15 min after kaolin injec- at room temperature with a rotating evaporator. Desic- tion in the control group were more than 12 (14.34±0.87, cated samples dissolved in 1.5 ml of distilled water were in 4 tests). Writhing responses attained a peak at 10 min diluted with buffer (6-keto-PGFIa EIA system; Amer- after kaolin injection and then attenuated at 15 min (Fig. sham Int. plc., Buckinghamshire, England), and 6-keto- 2). M-5011 inhibited kaolin-induced writhing responses PGFIa contents were determined by the enzyme im- in a dose-dependent manner (Table 1). The ED50 value munoassay method (6-keto-PGFIa EIA system) based on of M-5011 (0.63 (0.26-1.54, 95% CL) mg/kg) against a competitive reaction of enzyme-labeled 6-keto-PGFIa in writhing was 7.6- to 26.7-fold lower than that of TIA the samples for 6-keto-PGFIa antibodies. The lower and (4.78 (1.49-15.33) mg/kg) or ZLT (16.80 (7.52-37.51) upper detection limits of 6-keto-PGFIa were within 10- mg/kg), the same as that of DIF (0.68 (0.26-1.81) 1,280 pg/ml. Inhibition (%) was calculated as follows: Inhibition (%)=(A-B)/A x 100 where A=6-keto-PGFIa level in the control group and B=6-keto-PGFIa level in the drug-treated group.

Ulcerogenic test According to the report by Imayoshi et al. (11), with a slight modification, groups of 10 ddY mice (5 weeks) each were administered orally with M-5011 (168.75, 112.50, 75.00, 50.00, 33.33, 22.22 or 14.81 mg/kg); IND (20.00, 13.33, 8.89, 5.93, 3.95 or 2.63 mg/kg); KP (45.00, 30.00, 20.00, 13.33, 8.89, 5.93 or 3.95 mg/kg); DIF (8.89, 5.93, 3.95, 2.63, 1.76 or 1.17 mg/kg); TIA (168.75, 112.50, 75.00, 50.00, 33.33 or 22.22 mg/kg); ZLT (168.75, 112.50, 75.00, 50.00, 33.33 or 22.22 mg/kg) or vehicle before water and food were given ad libitum for 24 hr. Mice were then injected i.v. with 0.2 ml of saline solution containing 1% Evan's blue (Sigma Chem. Co.) before sacrifice by exsanguination. Stomachs and whole small intestines (from pyloric end to ileocecal part) were iso- lated, fixed by intraluminal irrigation with 2 ml of 1 % formalin solution (Nacalai Tesque) prior to ulceration analysis under microscopy. The incidence of ulceration, including ecchymosis, was evaluated on an all-or-none Fig. 2. Changes in writhing response after i.p. injection of kaolin suspension (2.5 mg in 0.5 ml of saline) containing 0.05 mg of cap- basis. topril in mice. The number of writhing responses were counted at 5-min intervals after kaolin injection. Each point represents the mean±S.E. (n=46). mg/kg), and 2.3- to 3.0-fold higher than those of IND that peritoneal BK was released by kaolin. Pretreatment (0.21 (0.14-0.31) mg/kg) and KP (0.28 (0.17-0.49) (1 hr before kaolin injection) with all drugs (M-5011, mg/kg) (Table 1). IND, KP and DIF: 3.0 mg/kg; TIA: 10.0 mg/kg; or ZLT 30 mg/kg) did not affect peritoneal BK production at 5 Effects of NSAIDs on BK levels induced by kaolin injec- min post-injection of kaolin with the indicated doses tion (data not shown). Kaolin-induced BK production in the peritoneal cavity from 3 to 10 min post-injection (Fig. 3). Peritoneal BK Effects of NSAIDs on kaolin-induced 6-keto-PGFIa levels in the control group peaked at 5 min post-injection production of kaolin (7.60± 1.83 ng/mouse) and decayed gradually Kaolin-induced 6-keto-PGFIa production in the thereafter. Similar treatment with physiological saline peritoneal cavity from 5 to 15 min post-injection (Fig. 3). (0.5 ml) did not induce any BK production, indicating Peritoneal 6-keto-PGFIa contents in the control group

Test compounds were administered orally 1 hr before i.p. injection of kaolin suspension (2.5 mg in 0.5 ml of saline) containing 0.05 mg of captopril. Each group consisted of 10-14 mice. The ED50 values and 95% confidence limits (CL) were calculated by regression analysis using inhibition per- centage. Potency ratios of drugs were calculated with indomethacin =1. Significant difference from the control, *P<0.05, **P<0.01. peaked at 10 min (16.54±3.04 ng/mouse) and decayed thereafter. Similar treatment with physiological saline (0.5 ml) did not induce any 6-keto-PGFIa production (less than 0.12 ng/mouse), implicating that peritoneal 6-keto- PGFIa was dependent on kaolin injection. Pretreatments with M-5011 and other NSAIDs (1 hr before kaolin injec- tion) elicited dose-related decreases in peritoneal 6-keto- PGFIa contents at 7.5 min post-injection of kaolin, co- inciding well with the peak number of kaolin-induced writhing responses. The ID50 value of M-5011 (0.66 (0.37-1.17, 95% CL) mg/kg) against kaolin-induced 6-

Fig. 3. Changes of both bradykinin (0) and 6-keto-PGFIa (•) contents in peritoneal fluid after i.p. injection of kaolin suspension (2.5 mg in 0.5 ml of saline) containing 0.05 mg of captopril in mice. Each point represents the mean ±S.E. (n=4-5).

Table 2. Effects of M-5011 and other non-steroidal anti-inflammatory drugs on increased levels of 6-keto-PGFIa induced by intraperitoneal injection of kaolin in mice

Test compounds were administered orally 1 hr before i.p. injection of keolin suspension (2.5 mg in 0.5 ml of saline) containing 0.05 mg of captopril. Animals were sacrificed to collect peritoneal fluids samples at 7.5 min after keolin injection. Each group consisted of 8-13 mice. The ID50value and 95 o confidence limits (CL) were calculated by regression analysis using inhibition percentage. Potency ratios of drugs were calculated with indomethacin = 1. The ID50 value of ketoprofen was not ob- tained. Significant difference from the control, *P <0.05, **P <0.01. Fig. 4. Acute ulcerogenic activities of M-5011 and other non-steroidal anti-inflammatory drugs on both the stomach (A) and small intestine (B) in mice. Tests compounds were administered orally 24 hr before sacrifice. Stomachs and whole small intes- tines (from pyloric end to ileocecal part) were isolated prior to ulceration analysis under microscopy. The incidence of ulcera- tion, including ecchymosis, was evaluated on an all-or-none basis. 0: M-5011 (M), A: Indomethacin (I), /: Ketoprofen (K), 0: Diclofenac sodium (D), A: Tiaprofenic acid (T), A: Zaltoprofen (Z). Each symbol represents the mean ulceration incidence (°lo) (n=10).

keto-PGFIa production was 2.5- to 15.4-fold lower than (88.23 (70.15 -115.50, 95% CL) mg/kg) in the gastric ul- those of DIF (1.64 (0.57-4.74) mg/kg), TIA (2.79 cerogenicity was almost the same as those of ZLT (110.92 (1.61-4.83) mg/kg) and ZLT (10.18 (5.93-17.47) (86.87-156.05) mg/kg) and TIA (62.86 (49.68-79.78) mg/kg), but higher than that of IND (0.16 (0.11-0.24) mg/kg) (Table 3). Furthermore, M-5011 was respectively mg/kg). The ID50 value of KP was not calculated because 4.4-, 9.8- and 21.1-fold less potent than KP (20.04 the coefficient linearity by the three KP doses in the (15.87-25.98) mg/kg), IND (8.96 (7.12-11.57) mg/kg) present study could not be obtained (Table 2). and DIF (4.19 (3.28 - 5.52) mg/kg) in gastric ulcerogenic- ity. Effects of NSAIDs on gastric mucosa in mice The vehicle (CMC) did not produce any stomach ul- Effects of NSAIDs on small intestinal mucosa in mice cers. All drugs (including M-501 1) produced dose-depend- In a similar tendency, all drugs induced small intestinal ent ulceration (Fig. 4A). The UD50 value of M-5011 ulceration in a dose-dependent manner (Fig. 4B). The Analgesic safety index was defined as the ratio of UD50 to ED50 values of the writhing response.

UD50 values of NSAIDs on small intestinal ulceration for and other NSAIDs on the production of PGI2 or BK in M-5011, IND, KP, DIF, TIA and ZLT were 46.09 kaolin-induced writhing. (36.79-57.20), 4.78 (3.90-5.82), 10.75 (8.75-13.26), In the present study, an intraperitoneal injection of 2.24 (1.71-2.82), 46.55 (36.88-57.75) and 54.78 kaolin in mice caused clear and reproducible writhing (44.63 - 66.40) mg/kg, respectively (Table 3). reactions attaining a peak at 5 -10 min after kaolin injec- tion in agreement with the report of Fujiyoshi et al. (8). M-5011 and other NSAIDs elicited dose-dependent sup- pressive effects on kaolin-induced writhing. Based on It is well-known that substances with a negative surface ED50 values derived from these results, the potency rank charge such as kaolin activate the kallikrein-kinin system order of the drugs was as follows: IND ? KP > M-5011 through activation of blood coagulation factor XII (12). >_DIF > TIA > ZLT . Furthermore, at the peak of the In consequence, activation of the plasma kallikrein-kinin writhing responses, these drugs significantly reduced the system induces the generation of BK, causing potent pain peritoneal contents of 6-keto-PGFIa, a stable metabolite (13). Fujiyoshi et al. (8, 9) reported that the intra- of PGI2i in a dose-dependent manner, but not the peritoneal injection of kaolin produced kinin in the production of BK in agreement with previous findings peritoneal cavity of the mouse resulting in the writhing (12, 14). Thus, the potency rank of ID50 values of these reaction. Thus, it seems that kaolin-induced writhing is drugs on PGI2 production was similar to that of the anti- mainly based on BK. In addition to the effect of BK, it is nociceptive potency ranking of ED50 values. As a result, suggested that the sensory nerve-sensitizing effect of the present study indicates that the differences in the PGI2i an arachidonic acid metabolite, may play an im- antinociceptive potency of these acidic NSAIDs with portant role in the pain reaction in kaolin-induced writh- kaolin-induced writhing are at least partly based on the ing, from the evidence that this writhing reaction was potency of inhibition of PGI2 generation as stated in the markedly decreased by administration of acidic NSAIDs above findings (12, 14). such as IND that inhibit of cyclo-oxygenase (14). Fur- Apart from the antinociceptive potency of these acidic thermore, Joseph and Barry (15) and Hori et al. (16) NSAIDs accompanied by inhibition of PGI2 generation, reported that PGI2 was the most powerful substance to M-5011 displayed lower ulcerogenicity than IND, KP and cause a sensitizing potency of BK and acetic acid among DIF, and only TIA and ZLT, which had weak anti- the other arachidonic metabolites. Taken together, it nociceptive effects, showed lower ulcerogenicity. As a seems reasonable to consider that both BK and PGI2 is result, M-5011 revealed the highest analgesic safety index closely related to kaolin-induced writhing. Therefore, in (UD50/ED50). Although the mechanisms underlying the the present study, we tried to clarify the effects of M-5011 ability of NSAIDs to damage the gastrointestinal mu- cosa are not completely understood, there are a few steroidal anti-inflammatory drugs and corticosteroids on car- reports suggesting that unwanted gastric lesions induced tilage metabolism in rheumatoid arthritis and osteoarthritis. 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