Nitroparacetamol exhibits anti-inflammatory and anti-nociceptive activity

Abstract

Nitroparacetamol (NCX-701) is a newly synthesized nitric oxide-releasing derivative of paracetamol. Following i.p. administration, nitroparacetamol inhibits carrageenan-induced hindpaw oedema formation (ED₅₀, 169.4 μmol kg⁻¹) and mechanical hyperalgesia (ED₅₀, 156 μmol kg⁻¹) in the rat. In contrast, the parent compound, paracetamol, exhibits no significant anti-oedema activity in this model (ED₅₀>1986 μmol kg⁻¹, i.p.) and is markedly less potent than nitroparacetamol as an inhibitor of carrageenan-mediated hyperalgesia (ED₅₀, 411.6 μmol kg⁻¹, i.p.). In a second model of nociception (inhibition of acetic acid induced abdominal constrictions in the mouse), nitroparacetamol administered orally (ED₅₀, 24.8 μmol kg⁻¹), was again considerably more potent than paracetamol (ED₅₀, 506 μmol kg⁻¹, p.o.). Thus, compared with paracetamol, nitroparacetamol not only exhibits augmented antinociceptive activity in both rat and mouse but, intriguingly, is also anti-inflammatory over a similar dose range.

Introduction

Paracetamol exhibits both analgesic and antipyretic activity and as such is frequently classified alongside the nonsteroidal anti-inflammatory group of drugs (NSAID). However, paracetamol differs from NSAID in a number of important ways. For example, unlike NSAID, paracetamol exhibits little or no anti-inflammatory activity in animals (Glenn et al., 1977; Seegers et al., 1981; Bianchi & Penerai, 1996) and very limited, if any, anti-inflammatory activity in man (Lokken & Skjelkbred, 1980). Furthermore, unlike NSAID, paracetamol usage is associated with minimal incidence of gastrointestinal damage (Stern et al., 1984).

We (al-Swayeh et al., 2000), and others (Elliot et al., 1995; Wallace et al., 1995), have reported that nitric oxide (NO) releasing derivatives of NSAID (so-called NO-NSAID) exhibit greater anti-inflammatory, antinociceptive and anti-thrombotic activity than the parent NSAID. Evidence has been presented that the augmented anti-inflammatory effect of NO-NSAID relates to the slow release of NO from these compounds although the precise mechanism(s) involved remain unclear (for review, see del Soldato et al., 1999).

With these observations in mind we have now evaluated the anti-oedema and anti-nociceptive activity of nitroparacetamol (NOP) which is a newly synthesized NO-releasing derivative of paracetamol.

Methods

Rats (male, Wistar, 100–150 g, Tucks Ltd., U.K.) and mice (male, LACA, 22–35 g, Tucks Ltd., U.K.) were purchased from accredited suppliers and maintained in the Biological Services Unit of this College until required. All experiments were carried out ‘blind' in that the observer was not aware of the identity or dose of drugs administered to individual animals and were conducted under the conditions of the Animals Scientific Procedures Act, U.K. (1986).

Measurement of carrageenan-induced hindpaw oedema formation in the rat

Experiments were conducted as described previously (al-Swayeh et al., 2000). Briefly, hindpaw volume (ml) was determined by plethysmography before, and at timed intervals (30 min–6 h), after intraplantar carrageenan injection (100 μl, 2% w v⁻¹). Nitroparacetamol (25–100 mg kg⁻¹), paracetamol (2.5–300 mg kg⁻¹) or vehicle (0.5% w v⁻¹ carboxymethylcellulose, CMC, 1 ml kg⁻¹) were administered i.p. 15 min prior to carrageenan injection. At the end of the experiment, animals were killed by cervical dislocation and exsanguination. Results show mean±s.e.mean, n=5–24.

Measurement of carrageenan-induced hindpaw hyperalgesia

Experiments were conducted as described previously (al-Swayeh et al., 2000). ‘Baseline' measurements of hindpaw (injected and contralateral i.e. non-injected) mechanical nociceptive threshold were determined using a commercially available algesiometer (Ugo Basile Ltd.). Thereafter, hindpaw inflammation was induced by intraplantar injection of carrageenan (150 μl, 2% w v⁻¹) into either hindpaw chosen at random. Nitroparacetamol or paracetamol (25–100 mg kg⁻¹, i.p.) or an appropriate volume (5 ml kg⁻¹, i.p.) of vehicle (0.5% w v⁻¹ CMC) were administered 2.5 h after carrageenan injection and the mechanical nociceptive threshold determined 30 min thereafter. Results (mean±s.e.mean, n=10–15) are shown as the difference in nociceptive threshold (arbitrary units) before (‘pre-') and 3 h after (‘post-') intraplantar injection of carrageenan. Statistical comparisons between groups was determined as the difference in ‘post-pre' values.

Acetic acid-induced abdominal constriction assay

Experiments were conducted as described previously (Moore et al., 1991). Mice were administered nitroparacetamol, paracetamol (2.5–100 mg kg⁻¹, p.o.) or vehicle (0.5% w v⁻¹ CMC; 10 ml kg⁻¹, p.o.) 15 min prior to i.p. acetic acid (2% w v⁻¹ in saline; pH 2.7; 10 ml kg⁻¹). Animals were transferred immediately to individual observation cages and the number of abdominal constrictions monitored over the following 30 min. At the end of the observation period animals were killed by cervical dislocation and exsanguination Results show number of abdominal constrictions per 30 min test period and are mean±s.e. mean (n=10).

Statistics

The statistical significance of differences between groups was determined by ANOVA followed by post hoc Dunnett' test. A probability (P) value of 0.05 or less was taken to indicate statistical significance.

Materials

Nitroparacetamol (NCX-701; 4-(nitroxy)butanoic acid 4-acetylaminophenyl ester) was obtained from NiCOX Ltd. (Sophia-Antipolis, France). Paracetamol and carrageenan were purchased from Sigma Ltd. Paracetamol and nitroparacetamol were suspended in 0.5% w v⁻¹ carboxymethylcellulose (CMC). Carrageenan was suspended in saline. A reasonable suspension of all drugs was achieved after warming (45°C) and with constant sonication and agitation.

Results

Anti-inflammatory activity

Pretreatment with paracetamol (up to 300 mg kg⁻¹, i.p.) did not affect carrageenan-induced hindpaw oedema (P>0.05, ANOVA plus Dunnett' test) at any time point up to 6 h after carrageenan injection (Figure 1A). In marked contrast, nitroparacetamol administration resulted in a pronounced, dose- and time-related inhibition of carrageenan-induced hindpaw oedema (Figure 1B). The ED₅₀ value for nitroparacetamol (calculated at 180 min) was 47.8 mg kg⁻¹ (equivalent to 169.4 μmol kg⁻¹). For comparison, paracetamol (300 mg kg⁻¹, equivalent to 1986 μmol kg⁻¹) reduced oedema by 31.8% (P>0.05) at 180 min after intraplantar carrageenan injection.

Figure 1.

Rats were administered paracetamol (A) or nitroparacetamol (B) 15 min before intraplantar carrageenan. Doses shown are mg kg⁻¹ (i.p.). Hindpaw oedema (ml) was measured by plethysmography. Results shown mean±s.e.mean, n=5–24, ^*P<0.05.

Antinociceptive activity

In control, vehicle-injected mice, i.p. administration of acetic acid caused 52.1±2.7 (n=10) constrictions over the 30 min observation period. Pretreatment of animals (30 min before acetic acid injection) with paracetamol resulted in dose-related inhibition of abdominal constrictions (Figure 2A). The dose required for significant antinociceptive activity was 100 mg kg⁻¹ (equivalent to 720 μmol kg⁻¹). Thus, it may be concluded that paracetamol exhibits modest antinociceptive activity in this model. Again, in marked contrast, nitroparacetamol caused dose related inhibition of acetic acid-induced abdominal constrictions (Figure 2B). In this case, the dose required for threshold antinociceptive activity was 2.5 mg kg⁻¹ (equivalent to 8.9 μmol kg⁻¹) whilst the ED₅₀ was 7 mg kg⁻¹ (equivalent to 24.8 μmol kg⁻¹). Thus, the potency ratio (nitroparacetamol vs paracetamol) is calculated to be 20.3 on a mol for mol basis.

Figure 2.

Inhibition of acetic acid induced abdominal constrictions in the mouse by paracetamol (A) and nitroparacetamol (B). Doses shown are mg kg⁻¹ (i.p.). Control animals received an appropriate volume of vehicle. Results shown mean±s.e.mean, n=10, ^*P<0.05.

In separate experiments, intraplantar administration of carrageenan (2% w v⁻¹, 150 μl) in the rat caused significant hindpaw hyperalgesia as evidenced by a reduction in the threshold for noxious mechanical stimulation from 9.92±1.0 units to 3.38±0.37 units (n=6, P<0.05). Pre-treatment of animals (150 min after intraplantar carrageenan injection; i.e. 30 min before test) with paracetamol or nitroparacetamol (25–100 mg kg⁻¹, i.p.) inhibited carrageenan-induced hyperalgesia in the injected hindpaw (Figure 3). The calculated ED₅₀ values for paracetamol and nitroparacetamol as inhibitors of mechanical hyperalgesia are 62.2 and 44.0 mg kg⁻¹ (equivalent to 411.6 and 156 μmol kg⁻¹), respectively.

Figure 3.

Inhibition of mechanical hyperalgesia by paracetamol and nitroparacetamol. Drugs (mg kg⁻¹, i.p.) were administered 2.5 h after intraplantar carrageenan injection. Results show mean±s.e.mean, n=8.

Discussion

In the present study, pretreatment of animals with paracetamol caused dose related antinociceptive activity following either i.p. (rat) or p.o. (mouse) administration employing either carrageenan or acetic acid respectively as the nociceptive stimulus. The antinociceptive effect of paracetamol was apparent only at relatively high doses which lack of potency agrees with previous published reports on this compound. For example, Vinegar and colleagues (1976) noted that pretreatment with paracetamol inhibited acetic acid-induced abdominal constrictions in the mouse at a threshold dose of 200 mg kg⁻¹. Similarly high doses of paracetamol were also effective in other animal models e.g. zymosan-induced writhing (Doherty et al., 1990; threshold for antinociceptive activity, 200 mg kg⁻¹) as well as formalin-induced hindpaw licking in the mouse (Hunskaar & Hole, 1987) and rat (Kanui et al., 1993).

In separate experiments, paracetamol was devoid of significant anti-inflammatory activity (doses up to 300 mg kg⁻¹, i.p.). A similar lack of anti-inflammatory activity of paracetamol has also been noted by other researchers using similar animal models (e.g. Bianchi & Penerai, 1996; Glenn et al., 1977; Seegers et al., 1981).

With these observations in mind it is clearly of interest that nitroparacetamol not only exhibits greater antinociceptive activity than paracetamol with potency ratios (mol for mol basis) ranging from three (carrageenan-induced hindpaw hyperalgesia) to 20 (acetic acid induced abdominal constrictions) but also reduces carrageenan-induced hindpaw oedema formation at doses as low as 25 mg kg⁻¹ (i.e. at least 20 times more potent than paracetamol).

The mechanism(s) underlying the enhanced anti-inflammatory and antinociceptive activity of nitroparacetamol have not been investigated in the present experiments. Nevertheless, some comment appears warranted.

The limited antinociceptive as well as the almost complete lack of anti-inflammatory activity of paracetamol has frequently been ascribed to the (at best) very weak cyclo-oxygenase (COX) inhibitory activity of this compound (Abdel-Halim et al., 1978; Bippi & Frolich, 1990; Seppala et al., 1983).

Over the last several years there has been considerable debate in the literature concerning the possibility of a ‘cross talk' between COX, nitric oxide synthase (NOS) and their respective products. In the present experiments, it may reasonably be assumed that NO release from nitroparacetamol accounts for, or at least makes a major contribution to, the augmented biological activity of this compound. Thus, an inhibitory effect of NO on COX activity may be considered as a possible rationale for the increased biological activity of nitroparacetamol. In this context, NO donors such as S-nitroso-acetylpenicillamine have previously been reported to inhibit COX enzyme activity in vitro (e.g. Kosonen et al., 1998) whilst peroxynitrite inactivates prostacyclin synthase by nitration of strategically positioned tyrosine residues in the enzyme (Zou & Ullrich, 1996). However, it should also be noted that, under some conditions, NO may activate (not inhibit) COX enzyme activity (Salvemini et al., 1993). Thus, the precise interaction between NO (released from nitroparacetamol) and COX activity remains unclear. Other possible mechanisms of the anti-inflammatory effect of nitroparacetamol-derived NO include, (i) inhibition of caspase-1 (interleukin converting enzyme) and thus reduced formation of pro-inflammatory interleukin-1β (Dimmeler et al., 1997) and, (ii) inhibition of cellular transduction mechanisms underlying inflammation (e.g. NFκ-B cascade) (Minto et al., 1997). In this context, it is of interest that pretreatment of endotoxin-treated animals with nitroflurbiprofen has been reported to reduce iNOS induction in the stomach (Mariotto et al., 1995). Clearly, further experiments to examine the mechanism of the anti-inflammatory effect of nitroparacetamol are required.

Abbreviations

CMC

carboxymethylcellulose

COX

cyclo-oxygenase

NO-NSAID

nitro non-steroidal anti-inflammatory drug

NSAID

non-steroidal anti-inflammatory drug