British-Joumal of Phannacology (1995) 11A 303-308

h 1995 Stockton Press All rights reserved 0007-1188/95 $9.00

S14080, a peripheral analgesic acting by release of an endogenous circulating opioid-like substance

'Sergio H. Ferreira, Berenice B. Lorenzetti, *Michelle Devissaguet, tDaniel Lesieur & *Yannis Tsouderos
'Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, Ribeirao Preto, S.P., 14049-900, Brazil; *Institut de Recherches Internationales Servier, Courbevoie, 92415, France and tInstitut de Chimie Pharmaceutique, Lille, 59000, France
1 The oral administration of a benzothiazolinone derivative (benzoyl-6 dihydro-2,3 benzothiazole), S14080, caused dose-dependent antinociception in the rat paw pressure test, which represents a model of mechanical hyperalgesia. S14080 had no significant effect on the inflammatory oedema induced by carrageenin or on the tail flick test, nor did it possess a notable antipyretic effect. 2 Post-treatment with S14080 dose-dependently antagonized the hyperalgesia induced by prostaglandin E2, bradykinin, dopamine and by the hyperalgesic cytokines reported to be released by carrageenin (tumour necrosis factor a, interleukin-l and interleukin-8). 3 The blockade of prostaglandin E2-induced paw hyperalgesia by oral pretreatment of the rats with S14080 was abolished by prior intraplantar administration of either naloxone or NorBNI which are non-specific and specific K opioid antagonists, respectively. 4 Adrenalectomy abolished the oral antinociceptive effect of S14080. 5 Five consecutive daily injections of S14080 did not produce tolerance such as that seen with the central antinociceptive action of morphine. 6 As with peripherally acting opiates, the antinociceptive activity of S14080 was abolished by the intraplantar injection of agents which inhibit either arginine synthetase (N0-monomethyl-L-arginine) or the activation of guanylate cyclase (methylene blue). 7 We conclude that S14080 is a new type of peripheral antinociceptive which, in rats, acts mainly by releasing an endogenous, opioid-like substance from the adrenal glands. Keywords: Peripheral opioid antinociception; S14080 antinociception; adrenal glands antinociception; peripheral analgesia; endogenous opioid antinociception

Introduction
Hyperalgesia is a fundamental event in the manifestation of overt, inflammatory pain. Peripheral antinociceptives which interfere with inflammatory hyperalgesia can be divided into two broad groups: (a) those which prevent the sensitization of pain receptors (Ferreira, 1972; Ferreira et al., 1973; Ferreira, 1980), e.g. cyclo-oxygenase inhibitors (non-steroidal anti-inflammatory drugs) and (b) those which directly abolish the ongoing sensitization of pain receptors, e.g. dipyrone and peripheral opioids (Ferreira & Nakamura, 1979; Lorenzetti & Ferreira, 1982; 1985; Ferreira et al., 1984; Ferreira, 1990). There are, however, drugs in the second group which may act as antinociceptives, i.e., by blocking ongoing hyperalgesia, through the peripheral release of an endogenous, opioid-like substance. We have found that clonidine and St-91 (a clonidine analogue which does not cross the blood-brain barrier) cause peripheral antinociception which is abolished by the intraplantar administration of opioid antagonists (Nakamura & Ferreira, 1988). The clonidine antinociceptive activity was blocked by yohimbine, an M2-adrenoceptor antagonist. General screening of S14080, a benzothiazolinone (benzoyl-6 dihydro-2,3, benzothiazole (Follet-Houttemane et al., 1991), revealed a marked antinociceptive effect on acetic acid-induced writhing in the mouse. In the present paper, we report investigations into the mechanism of this antinociceptive action of S14080. This substance was initially tested in the rat paw hyperalgesia assay (Ferreira et al., 1978) employing carrageenin and mediators thought to be released by carregeenin such as prostaglandin E2 dopamine, interleukin-l (IL-1) and IL-8, as nociceptive stimuli. We have also comI

pared the antinociceptive effect of S14080 with that of typical cyclo-oxygenase inhibitors (indomethacin) and of compounds which counteract ongoing hyperalgesia induced by prostaglandin E2 (dipyrone). The antinociceptive profile of S14080 was not similar to that of indomethacin or dipyrone. Based on the abolition of its effect by adrenalectomy and opioid antagonists, S14080 appears to cause peripheral antinociception via the release of an endogenous opioid from the adrenal glands. In contrast to clonidine, this effect was not blocked by yohimbine. The antinociceptive activity of S14080, like that of dipyrone and peripheral opioids, was abolished by methylene blue, an inhibitor of guanylate cyclase activation, and by N0-monomethyl-L-arginine (L-NMMA), an inhibitor of the formulation of NO from L-arginine (Duarte et al., 1991a,b; Ferreira et al., 1991). The maximal effect of oral administration of S14080 was observed with 1 h of administration and no tolerance was observed following five days of repeated administration. These results suggest that S14080 constitutes a new type of peripheral antinociceptive whose mode of action involves the release of an endogenous opioid-like substance.

Methods
Animals
Male Wistar rats (130-180 g) with free access to food and water were used for the various experiments. When the drug was administered orally, the animals were deprived of food 12 h before testing although water was allowed ad libitum.

Author for correspondence.

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A peripheral oploid-like analeic

Nociceptive test
Hyperalgesia was measured by our modification (Ferreira et al., 1978) of the Randall-Selitto (Randall & Selitto, 1957) test. In this test, a constant pressure of 20 mmHg is applied to the rat paw and discontinued (reaction time) when the animal exhibits a reaction characterized by the reduction of escape movements followed by an alteration in the respiratory frequency and the onset of a typical shivering reaction. The intensity of hyperalgesia was quantified as the difference in reaction reduction time (A reaction time) obtained by subtracting the value measured 2 or 3 h after administration of the hyperalgesic substances from the control reaction time, measured prior to the administration of the hyperalgesic substance. Hyperalgesia was induced in the hind paws by the intraplantar (i.pl.) administration of 100 or 300 fig of carrageenin, 100 ng of prostaglandin E2, 500 ng of bradykinin, 0.1 ng of IL-8, 10 ;tg of dopamine, 0.1 i.u. of IL-1p or 2.5 pg of tumour necrosis factor-a (TNF-a), injected in 100 jl of saline. These substances have been shown to produce quantitatively equivalent levels of hyperalgesia. The investigator was unaware of the order of drug treatments applied.

NMMA, Weilcome, England), NorBNI (nor binaltorphimine dihydrochloride) (kindly provided by Dr K. Rice, NIH, U.S.A.), benzoyl-6 dihydro-2,3 benzothiazole (S14080, Institut de Rechereches Internationales Servier, France, dissolved in peanut oil from Sigma, U.S.A.), lipopolysaccharide (LPS) from E. coli 0111:B4, dopamine (3-hydroxytyramine hydrochloride) and bradykinin (Sigma, U.S.A.), yohimbine hydrochloride and morphine hydrochloride (Merck, Germany), IL-1p, IL-8 and TNFa (NIBSC preparations coded 86/680, 89/520 and 87/650 respectively), naloxone hydrochloride (Du Pont Pharmaceuticals, U.S.A.).

Results

The effect of oral adninistration of S14080 on hyperalgesia and oedema induced by two intraplantar injections (100 jig or 300 fig) of carrageenin Oral pretreatment with S14080, 10-40mgkg-' resulted in
dose-dependent antinociception for both low doses (ED50 = 17.5, c.l. 8.9-34.5 mg kg-') and high doses (ED50 = 41.0, c.l. 14.2-118.4 mg kg-') of carrageenin (Figure la,b; ANOVA, P<0.0001). In contrast to the antinociceptive effect on carrageenininduced hyperalgesia, S14080 in doses up to 80mgkg' had no antinociceptive action when assessed by the tail flick method (results not shown). This method detects the central effect of antinociceptive agents (Smith, 1965). There was no significant concomitant effect of S14080 on the accompanying oedema (Figure lc,d; ANOVA, P>0.5).

Oedema
The oedema induced by the i.pl. injection of carrageenin (100 and 300 pg) was measured plethysmographically (Ferreira, 1979). Oedema formation was defined as the difference in paw volume (jl) measured before and 2 or 3 h after i.pl. carrageenin administration.

Effect of S14080 on the hyperthermia induced by the intravenous injection of lipopolysaccharide (LPS) Rectal temperature was measured by inserting a z thermistor
probe (Y.S.I. nr. 401) 3 cm into the rectum. The animals were held gently during the temperature measurements. This procedure was performed at least twice, one day prior to the experiment in order to minimize pos$ible stress-induced temperature changes. The experiments were conducted during a single, one-day period. Basal temperature was defined by recording four measurements taken at 30 min intervals, before the administration of the drugs. The experiments were conducted in the thermoneutral zone for rats (28 1C) (Gordon, 1990).

Antinociceptive action of S14080 on various hyperalgesic stimuli

In this series of experiments, S14080 was tested against hyperalgesic mediators reported to be released by carrageenin in the rat (Ferreira et al., 1978; 1988; Nakamura & Ferreira, 1987; Cunha et al., 1991; 1992). Figure 2 shows that the oral administration of S 14080 produced dose-dependent antinociception after the application of prostaglandin E2 (PGE2), dopamine, bradykinin (BK), tumour necrosis factor a (TNF-x) or the recombinant interleukins-lp and 8 (IL-lp and IL-8). Control doses for the hyperalgesic substances were selected based on previous experiments in order to provide a qualitatively equivalent hyperalgesic response. The comparisons were made 3h after the challenge when the hyperalgesia induced by the various substances reached a plateau.

Presentation of the data and analysis The dose, route and time of administration of each substance are indicated in the figure legends. The results are presented as the mean s.e.mean and the statistical analysis employed indicated. No statistical test was applied when the difference between means was greater than three s.e.mean. Standard errors smaller than the symbols used are not shown. A one-way analysis of variance (ANOVA) was used to compare the changes between different doses or treatments. When a significant effect was indicated, comparisons between individual means were subsequently tested using the Bonferroni procedure (Graph Pad in Stat, version 1.0). Differences were considered to be significant at P < 0.05. The ED50 and 95% confidence limits (c.l.) were calculated according to Litchfield & Wilcoxon (1949).

Duration of antinociception following oral administration of S14080 S14080 (40mgkg-') administered at different times before the measurement of hyperalgesia induced by PGE2 caused a time-dependent antinociception. Hyperalgesia was induced by PGE2 (100ng/paw) and measurements were performed 3h later. In this experiment, total inhibition of the hyperalgesia induced by PGE2 alone was considered to be 100% (19.1 0.5 s). Antinociception was maximal (92.7%;

1.4 0.5 s) 1 h after oral S14080 administration and subsequently decreased with time, reaching less than 12% after 4 h (17.0 1.0).

Drugs
Unless otherwise stated, the drugs were diluted in sterile saline. The following drugs were used: carrageenin (Viscarin Marine Colloids, U.S.A.), prostaglandin E2 (Upjohn, U.S.A.), indomethacin (Merck, Sharp & Dohme, Brazil, dissolved in Tris-HCI buffer, pH 8.2), dipyrone and clonidine
blue

hydrochloride (Boehringer Ingelheim, Germany), methylene (Reagen, Brazil), NG-monomethyl-L-arginine (L-

Intraplantar effect of NorBNI on the systemic action of indomethacin, dipyrone and S14080 upon PGE2-induced hyperalgesia Figure 3 shows the effect of NorBNI (5 tg/paw) on the systemic action of S14080 (10, 20 and 40 mg kg-'), indomethacin (5 mg kg-') and dipyrone (20mg kg-') upon PGE2-induced hyperalgesia. Figure 3a shows that S14080 caused dose-dependent antinociception which was prevented

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Figure 2 Analgesic effect of S14080 on the hyperalgesia induced by different stimuli. The control (C) doses of the hyperalgesic stimuli were: prostaglandin E2 (PGE2, 100 ng), dopamine (DA, 10 jAg), bradykinin (BK, 500 ng), tumour necrosis factor (TNF-a, 2.5 pg), interleukin-lP (IL-IP, 0.1 UI) and interleukin 8 (IL-8, 0.1 ng). S14080 was administered 2 h after injection of the hyperalgesic stimulus. Data are the mean s.e.mean of five rats per group. The hyperalgesia was measured 3 h after injection of the stimuli.

306

6S.H. Ferreira et al

A peripheral opioid-like analgesic

by previous intraplantar injection of NorBNI; Figure 3b confirms earlier results (Lorenzetti & Ferreira, 1985) by showing that the administration of indomethacin or dipyrone resulted, respectively, in either the absence of antinociception or in intense antinociception on the hyperalgesic response to PGE2 (open columns). The intraplantar injection of the opioid antagonist NorBNI did not affect either control prostaglandin hyperalgesia or the responses to the treatment with dipyrone or indomethacin (hatched columns). Naloxone (Nx, 1 jg/paw and 1 mg kg-1) also abolished the action of S 14080 (data not shown).

measurable difference between the control and treated hyperalgesic groups (ANOVA, P <0.05, Figure 4). No variation in the antinociceptive effect was observed during the treatment with S14080 (40mg kg-'), (ANOVA, P<0.05).

Methylene blue and L-NMMA inhibit S14080-induced antinociception

Figure 5 shows the effect of pretreatment with methylene blue (MB) or L-NMMA on the antinociceptive action of S14080 in hyperalgesic, PGE2-treated paws. MB did not affect the hyperalgesia induced by PGE2 but abolished the antinociceptive effect of S14080. A similar response was observed in paws pretreated with L-NMMA.

Yohimbine does not inhibit the antinociceptive action of S14080

As previously shown (Nakamura & Ferreira, 1988), the antinociceptive effect of clonidine (250 lgkg-', i.p.) against PGE2-induced hyperalgesia (18.1 0.6 s) was blocked (5.7 0.4 s) by pretreatment with yohimbine, (1 mg kg-', i.p., 16.0 0.6 s). The pretreatment of the animals with yohimbine, however, had no significant effect (P> 0.05 Bonferroni) on the antinociceptive action of S14080 (10, 20, 40 mg kg-', orally).

S14080 does not prevent the febrile response induced by LPS in rats
Oral pretreatment with S14080 (40 and 80 mg kg-') 30 min before the injection of LPS, unlike indomethacin (8 mg kg-'),
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Adrenalectomy abolishes the oral antinociceptive potency of S14080

Sham operation did not alter the antinociceptive action of S14080 (40 mg kg-', p.o., 5.2 0.5 s) or of dipyrone (40 mg kg-', p.o., 5.2 0.4) on the hyperalgesia induced by prostaglandin E2 (18.8 0.5). Adrenalectomy, however, abolished the antinociception produced by S14080 (17.9 0.7), without influencing the response to dipyrone (5.2 0.7).

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Response injections of S14080 do not induce tolerance There was a significant difference compared to the effect of morphine after consecutive applications (ANOVA, P<0.05). Antinociceptive tolerance to centrally acting opioids such as morphine occurred following five, consecutive daily treatments (8 mg kg-'). After this period, there was no
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Figure 4 Lack of tolerance to S 14080 as compared with morphine, a centrally acting opiate. S14080 (A, 40 mg kg-') and morphine (0, 8 mg kg-') were given orally, two times a day for five consecutive days. Prostaglandin (PGE2) (0, 100 ng/paw) was injected in alternate paws 2 h before the first daily injection of the analgesics. Data are the mean s.e.mean of five animals per group. The intensity of hyperalgesia was measured 3 h after the PGE2 challenge.

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Figure 3 NorBNI, an opioid K-antagonist, inhibits the analgesic action of S 14080, but not that of indomethacin or dipyrone, in prostaglandin E2 (PGE2)-induced hyperalgesia. (a) Shows the analgesic effect of increasing doses of S14080 (0) on the hyperalgesia induced by PGE2 (100 ng/paw). A single dose of NorBNI (5 pg/paw, 0) was sufficient to inhibit this action of S14080. (b) Shows that NorBNI (cross-hatched columns), in the same dose as for (a) had no influence on the responses to indomethacin (Ind, 5 mg kg-') or dipyrone (DP, 20 mg kg-') in PGE2-induced (100 ng/paw) hyperalgesia. Note that NorBNI had no effect by itself on PGE2-induced hyepralgesia (time 0 on (a) and open column C in (b)). In both (a) and (b), NorBNI and the analgesics were injected 90min and 120min, respectively, after PGE2 application. Data
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Figure 5 Methylene blue and N'-monomethyl-L-argiine (L-NMMA) inhibit the analgesic action of S14080. Hyperalgesia was induced by the injection of prostaglandin E2 (PGE2, C, 100Ong/paw). Ninety minutes later, the animals were treated with methylene blue (MB, 500 jig/paw) or L-NMMA (50 ILg/paw). SI14080 (SI, 40 mg kg-') was administered orally 2 h after the prostaglandin challenge. The intensity of hyperalgesia was measured 3 h after the PGE2 challenge. Data are the mean s.e.mean of five animals per group.

S.H. Ferreira et al
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Time (h) 6 The effect of pretreatment with S14080 on the febrile Figure response induced by lipopolysaccharide (LPS) in rats. Indomethacin (8mg kg-') and S14080 (S14, 40 and 80mg kg-') were administered orally 30 min before the intravenous administration of LPS (50 pg kg-'). (0) Control; (@) indomethacin 8 mg kg-'; S14080 (A) 40 mg kg-', (A) 80 mg kg-'. Rectal temperature was measured at 30 min intervals but only the hourly presented. Data are the
mean s.e.mean of five to size animals per group.

had no significant antipyretic effect (P> 0.05). There was, however, a transient blockade (1 h after LPS) of the febrile response with highest dose of S 14080 (80 mg kg-', Figure 6).

Discussion
In the present study, we have demonstrated that S14080 produces dose-dependent antinociception but has an inconsistent effect on the inflammatory oedema induced in the rat paw by carrageenin. The antinociceptive effect of S14080 was maximal 1 h after oral administration and, unlike morphine, the animal did not show tolerance after five consecutive daily

treatments. Previous studies by our group have shown that several cytokines cause hyperalgesia in the rat paw via the local release of hyperalgesic mediators. IL-1 and IL-8-induced hyperalgesia is mediated by cyclo-oxygenase products or sympathetic mediators (Cunha et al., 1991; Ferreira et al., 1988). In carrageenin-induced hyperalgesia, the release of these cytokines is preceded by the liberation of TNF-ac (Cunha et al., 1992). Recently, we reported that the hyperalgesia induced by bradykinin in the rat paw was also mediated by the release of an array of these same hyperalgesic cytokines (Ferreira et al., 1993a, 1993b). In the present study, S14080 displayed a dose-dependent, antinociceptive effect on hyperalgesia, not only against carrageenin, but also against mediators such as bradykinin, prostagland E2, dopamine and hyperalgesic cytokines (IL-1, IL-8) which are thought to be released during the inflammatory reaction (Cunha et al., 1991). Unlike indomethacin (a cyclo-oxygenase inhibitor), the general antinociceptive activity of S14080 against the various substances tested is typical of antinociceptives such as dipyrone and peripheral opiates which directly antagonize ongoing hyperalgesia (Lorenzetti & Ferreira, 1982; 1985; Ferreira et
References
CUNHA, F.Q., LORENZETTI, B.B., POOLE, S. & FERREIRA, S.H. (1991). Interleukin 8 as a mediator of sympathetic pain. Br. J.

al., 1984). However, although S 14080 did act in a qualitatively similar manner to dipyrone (Lorenzetti & Ferreira, 1985), its antinociceptive effects were in fact antagonized by the intraplantar injection of either NorBNI or naloxone, which are K and non-specific opioid antagonists, respectively. Thus, the mechanism of action of S14080 seems to be different from that of either indomethacin or dipyrone but has similarities to a peripheral, opioid effect. The absence of an antinociceptive role for S14080 in the tail flick test is in line with the suggestion that the site of action of S14080 is not central (Smith et al., 1985). The action of S14080 does not, however, seem to be via a direct antinociceptive effect on peripheral nociceptors since, in contrast with the action of dipyrone, S 14080 antinociception was strongly inhibited by adrenalectomy. Overall, our results suggest that the major site of action of S14080 is the adrenal glands where it induces the release of an endogenous, opioid-like substance. Indeed, adrenal glands do contain a large quantity of endogenous opioids, particularly of enkephalin (North & Egan, 1983). We have already shown that clonidine causes peripheral antinociception through the release of an opioid-like substance (Nakamura & Ferreira, 1988). However, unlike clonidine, the antinociceptive effect of S14080 was not inhibited by yohimbine. Thus, S14080 appears to be independent of M2-receptor activation which is associated with the hypotensive action of clonidine. We have suggested that clonidine-induced antinociception is not appropriate for clinical use since the antinociceptive action would probably be overshadowed by the hypotensive response, a phenomenon which would not be expected to occur with S14080. General screening of S14080 has shown no effect on rat blood pressure (unpublished results). We have previously demonstrated that various antinociceptives block hyperalgesia by stimulation of the arginine/NO/ cyclicGMP nociceptor pathway (Ferreira et al., 1991; Duarte et al., 1992). In line with this conclusion, we have shown here that S14080-mediated antinociception was also abolished by the intraplantar injection of agents which inhibit the arginine/nitric oxide/cyclicGMP pathway, namely L-NMMA, an inhibitor of NO synthetase, and methylene blue, an inhibitor of the activation of guanylate cyclase. S14080 in doses which cause significant antinociception had an inconsistent effect on carrageenin-induced oedema, and a negligible antipyretic effect. In the rat, the antipyretic and anti-oedematogenic activities are the two major characteristics of non-steriodal anti-inflammatory drugs which inhibit cyclo-oxygenase. This suggests that the oral administration of S14080 has little or no effect on tissue cyclooxygenase. Alternatively, a poor distribution of S14080 within the central nervous system may explain the absence of an antipyretic effect. Whatever the mechanism of action maybe, a peripheral antinociceptive without an antipyretic effect is a clinically desirable characteristic in the follow up of patients in which the absence of fever is a sign of good prognosis. In conclusion, S 14080 appears to be a new type of antinociceptive which acts peripherally via the release of an endogenous, opioid-like substance.
The authors thank Ms I.R. Santos for technical assistance. This work was supported by FAPESP and CNPq (Brazil).

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(Received May 31, 1994 Revised August 22, 1994 Accepted September 16, 1994)