Protective effect of the tachykinin NK2receptor antagonist nepadutant in acute rectocolitis induced by diluted acetic acid in guinea-pigs

Neuropeptides (2000) 34 (6), 355–359 © 2000 Harcourt Publishers Ltd doi: 10.1054/npep.2000.0819, available online at http://www.idealibrary.com on

Protective effect of the tachykinin NK2 receptor antagonist nepadutant in acute rectocolitis induced by diluted acetic acid in guinea-pigs C. Cutrufo, S. Evangelista, R. Cirillo, A. Ciucci, B. Conte, G. Lopez, S. Manzini, C. A. Maggi Menarini Ricerche spa, Pharmacology and Preclinical Development Departments, Firenze and Pomezia (Roma), Italy

Summary We have evaluated the potential protective activity of nepadutant, a selective tachykinin NK2 receptor antagonist, in a model of acute rectocolitis induced by an enema with 7.5% acetic acid in guinea-pigs. The injury was quantified visually by using a macroscopic injury score, and histologically by using a necrosis score. In addition, changes in myeloperoxidase activity, a marker for neutrophil infiltration, and plasma protein extravasation were evaluated. The injury caused by 7.5% acetic acid was mild, affecting the superficial layers and producing a strong edema of the submucosa. A single administration of nepadutant (0.3–10 mg/kg s.c., 1 h before acetic acid) markedly reduced the macroscopic damage and necrosis score and the increase in plasma protein extravasation induced by 7.5% acetic acid in the early phase of the injury. Single administration of nepadutant (3 mg/kg s.c.) reduced the macroscopic score and myeloperoxidase activity at the top (24 h) of inflammation. Repeated administration (3 mg/kg s.c. three times during 24 h) or co-administration of the tachykinin NK1 receptor antagonist MEN 11467 (3 mg/kg s.c.) did not enhance the antiulcer effect obtained with the single treatment with nepadutant. These data suggest the involvement of tachykinin NK2 receptors in the first phases of inflammation induced by acetic acid. © 2000 Harcourt Publishers Ltd

INTRODUCTION The role of tachykinins in the experimental models of inflammatory bowel diseases is matter of debate (Holzer 1998; Quartara and Maggi 1998). Tachykinins have been described to participate in several stages of the diseases and to influence the associated changes in motility, ion and fluid secretion and pain perception (Holzer 1998). A number of novel selective tachykinin receptor antagonists are now available which enable the precise assessment of the potential involvement of tachykinin receptors in these mechanisms of disease. We have recently developed in our laboratory an acute model of rectocolitis induced by diluted enema of acetic acid in the guinea-pig (Cutrufo

Correspondence to: Dr. Stefano Evangelista, Menarini Ricerche spa, Via Sette Santi 1, 50131 Firenze, Italy. Tel.: +39+055/5680519; Fax: +39+055/5680510; E-mail: [email protected]

et al. 1999). Acetic acid-induced injury is characterized by inflammation of the superficial gut layers, causing destruction of the mucosa, infiltration of inflammatory cells and submucosal edema. Unlike other chemical agents inducing colitis (e.g. trinitrobenzenesulphonic acid or TNBS), acetic acid produces a transient inflammation which does not progress into chronic inflammation. We found that the tachykinin NK1 receptor antagonist MEN 11467 markedly reduced the extent of tissue injury, possibly by inhibiting the acetic acid-induced increase in tissue permeability during the early phase of damage. On the other hand, the tachykinin NK2 receptor antagonist nepadutant has been recently shown to influence some aspects associated with the colitis formation. In fact it decreased the painful sensation induced by colorectal distention in stress or TNBS treated rats (Toulouse et al. 2000) and in this latter model reduced the noxious distention-induced c-Fos and c-Jun immunoreactivity in spinal cord and dorsal root ganglia, respectively (Kiss et al. 1999; Olivar et al. 1999). 355

356 Cutrufo et al.

In light of the above and of the reported effect of another tachykinin NK2 receptor antagonist in experimental ileitis induced in guinea-pig (Mazelin et al. 1998), we found it worthwhile to investigate the role of the NK2 antagonist nepadutant in diluted acetic acid induced rectocolitis. MATERIAL AND METHODS

with haematoxylin and eosin. The severity of mucosal necrosis was measured according to the following score: 1 = minimum: focal points of damage (change in architecture of the layers) in the whole section; 2 = slight: damaged areas in less than 20% of the whole section; 3 = moderate: damaged areas in 20 to 50% of the whole section; 4 = marked: damaged areas in 50 to 75% of the whole section; 5 = severe: damaged areas involving the whole section.

Animals Male Dunkin-Hartley guinea-pigs, 300–350 g body weight, were purchased from Charles River (Calco, Italy). Animals were fasted for at least 24 h before experiments. Induction of rectocolitis and evaluation of macroscopic and necrosis scores Guinea-pigs were anaesthetized with pentothal sodium (50 mg/kg s.c.). Twenty minutes later, a rubber cannula (8 cm) was inserted into the colon through the anus. Acetic acid (7.5%) was instilled intrarectally, the cannula was left in the colon for 30 seconds and then the animal was placed in its cage to recover from anaesthesia. The control group was treated with saline intrarectally. On the basis of previous results (Cutrufo et al. 1999), two times of sacrifice were chosen: 2.5 or 24 h after the enema of 7.5% acetic acid. Animals received nepadutant, MEN 11467 or both s.c. 1 h before the administration of 7.5% acetic acid, and in other experiments they received also supplementary treatments with nepadutant 6 and 23 h after the 7.5% acetic acid enema. For the evaluation of the tissue damage, each animal was killed with CO2, and the distal colon was excised (5–7 cm) from a fixed site. The damage in all specimens was immediately evaluated macroscopically and scored. Half of the specimen was then placed in Bouin’s solution (5% picric acid, 5% formalin and 1% acetic acid) for histological examination and the remaining half was rapidly frozen in liquid nitrogen and stored at –80°C for myeloperoxidase determination. Assessment of macroscopic changes of the colonic mucosa was performed according to McCafferty et al. (1994), with minor modifications. Macroscopic scores were assigned as follows: 0 = normal appearance, 1 = hyperaemia of the mucosa in less than 50% of the specimen, absence of macroscopic ulceration, 2 = strong hyperaemia of the mucosa extending throughout the specimen, bowel wall thickening, absence of macroscopic ulcerations, 3 = severe hyperaemia, with numerous areas of ulceration (size <0.5 cm), 4 = severe hyperaemia, with numerous areas of ulceration (size >0.5 cm). Histological examination of the tissue samples was performed as follows: after dehydration and embedding in paraffin, tissue sections (5 µm thickness) were stained

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Myeloperoxidase (MPO) activity Specimens of guinea-pig rectocolon (400–700 mg) were homogenized in 0.5% hexadecyltrimethylammonium bromide and 50 mM potassium phosphate buffer (pH=6.0), using a Polytron tissue homogenizer. After homogenization, the homogenizer was rinsed twice with 1 ml of hexadecyltrimethylammonium bromide buffer. The pooled homogenate and washes were sonicated for 15 seconds and the released enzyme was separated from insoluble cellular debris by centrifugation at 3000 g for 10 min. Myeloperoxidase activity was measured spectrophotometrically: 0.1 ml of supernatant was added to 2.9 ml of 50 mM potassium phosphate buffer, pH=6.0, containing 0.167 mg/ml o-dianosine hydrochloride and 0.0005% hydrogen peroxide. The change in absorbency at 1 and 5 min was measured with a spectrophotometer (Beckman DU-7, wavelength=460 nm). One unit of myeloperoxidase activity is defined as the amount able to degrade 1 µmol of peroxide per minute at 25°C. Evaluation of plasma protein extravasation of guineapig rectocolon In separate experiments the animals were surgically prepared according to Cutrufo et al. (1999) and Blue Evans was administered intravenously (20 mg/kg in saline containing 210 I.U./2 ml heparin) and then, 10 min later, the animals were perfused via the left ventricle with saline 100 ml/10 min to wash the dye out of the vasculature. The rectocolon was excised, weighed and the dye was extracted in 2 ml of formamide (50°C for 24 h). The amount of Evans blue was determined by spectrophotometry (wave length = 620 nm) and expressed as ng/mg of wet tissue. The amount of non-specific dye leakage in guinea-pig rectocolon was 10.6±0.1 ng Evans blue/mg tissue (n = 4). This value was subtracted from the plasma protein extravasation data presented in results, tables or figures. Drugs Nepadutant or cyclo{[Asn(β-D-GlcNAc)-Asp-Trp-Phe-DapLeu]cyclo(2β-5β)} was synthesized at the Chemistry

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Effect of tachykinin NK2 receptor antagonist nepadutant in acute rectocolitis 357

Department, Menarini Ricerche spa (Firenze, Italy) by conventional solid-phase method, dissolved in saline and administered in a volume of 5 ml/kg. MEN 11467 or (1R,2S)-2-N[1(H)indol-3-yl-carbonyl]-1-N{N-(p-tolylacetyl)-N-(methyl)-D-3(2-Naphthyl)alanyl}diaminocyclohexane) was synthetized at the Chemistry Department, Menarini Ricerche (Pomezia, Italy), dissolved in saline containing 2% Tween 80 and administered in a volume of 4 ml/kg. Acetic acid (Sigma) was diluted in saline to 7.5% concentration and administered intrarectally in a volume of 0.3 ml/animal. Pentothal sodium (Abbott, Italy) was dissolved in saline and given in a volume of 1 ml/kg. Statistics All data in the text are means ± SEM. Statistical significance was evaluated by means of one-way analysis of variance followed by Dunnett’s test. RESULTS Effects of nepadutant on acid acetic-induced injury (sacrifice at 2.5 h after 7.5% acetic acid enema) The microscopy studies indicate that of 7.5% acetic acid injury is characterized by the complete damage of the mucosal architecture and the epithelial layer with limited infiltration of inflammatory cells. In the submucosa there was marked edema which caused detachment of the layers. As shown in Table 1, 7.5% acetic acid produced remarkable macroscopic and necrosis scores matched by an increase in plasma protein extravasation, when compared to the saline treatment. At this sacrifice time there was a small increase in MPO activity. Pretreatment with nepadutant (1 h before the 7.5% acetic acid enema) produced a significant inhibition of macroscopic score, the values of percentage of inhibition being 5, 68 and 79 at

doses of 0.3, 3 and 10 mg/kg s.c., respectively(Table 1). The necrosis score induced by 7.5% acetic acid enema was significantly affected only by the higher dose of nepadutant (10 mg/kg s.c.) but the inhibition was 29, 32 and 68% with the doses of 0.3, 3 and 10 mg/kg s.c., respectively (Table 1). Plasma protein extravasation induced by the ulcerogen was markedly reduced (by 46%) by the pretreatment with 3 mg/kg s.c. of nepadutant (Table 1). Effects of nepadutant on acid acetic-induced injury (sacrifice at 24 h after 7.5% acetic acid enema) As shown in Figure 1, single administration of nepadutant, at the dose (3 mg/kg s.c.) shown to affect the injury and the tissue plasma protein extravasation during the early phases of the acetic acid inflammation, significantly reduced both the macroscopic damage score and MPO activity. The same dose of the tachykinin NK1 antagonist MEN 11467 was able to reduce the macroscopic score but not to affect the MPO activity (Fig. 1). The combined administration of the two tachykinin antagonists did not further increase the inhibitory values obtained with the single administration of nepadutant (Fig. 1). The effect on both parameters was not different if nepadutant was administered in single or repeated administration (–1, +6 and +23 h from the ulcerogen; Fig. 1).

DISCUSSION Acetic acid injury in the guinea-pig rectocolon provides a model of transient inflammation which does not progress into chronic inflammation; the present data indicate that it was reduced by the prior administration of the selective tachykinin NK2 receptor antagonist nepadutant. The effect should be ascribed to the NK2 antagonism being the affinity of nepadutant for the NK2 receptor 3–4 order

Table 1 effect of nepadutant on macroscopic damage score, necrosis score and plasma protein extravasation (PPE) in 7.5% acetic-induced rectocolitis. The animals were killed at 2,5 h after the 7.5% acetic acid enema. Group

Dose

n

(mg/kg)

Macroscopic

Necrosis

score

score

n

PPE (ng Blue Evans/ mg tissue

Saline

ñ

6

0±0

0±0

10

12± 2

Acetic acid

ñ

14

1.9 ± 0.06

3.1 ±0.3

14

37± 5a

Acetic acid+

0.3 s.c.

6

1.8 ± 0.1

2.2±0.7

5

31± 5

3 s.c.

8

2.1±0.3

7

20± 2b

10 s.c.

8

1.5 ±0.5c

ñ

n.t.

Nepadutant Acetic acid+

0.6 ± 0.1c

Nepadutant Acetic acid+

0.4 ± 0.1c

Nepadutant a

= P < 0.01 vs saline group; b = P < 0.05; c = P < 0.01 vs acetic acid group n.t. = not tested

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Neuropeptides (2000) 34(6), 355–359

358 Cutrufo et al.

4

12 10

3 MPO (U/ g)

2.5 2 1.5

8 6 4

1 2

0.5 0

AA+nep 3× 3

AA+nep+ MEN11467 3× 1

AA+MEN11467 3× 1

AA+nep 3× 1

AA+nep 3× 3

AA+nep+ MEN11467 3×1

AA+MEN11467 3×1

AA+nep 3× 1

AA+saline

0 AA+saline

Macroscopic score

3.5

Fig. 1 Effect of single (3 mg/kg s.c. once 1 h before acetic acid; 3×1) administration of nepadutant (nep), MEN 11467 or both or repeated (3 mg/kg s.c. three times 1 h before and 6 and 23 h after acetic acid; 3×3) administration of nepadutant on macroscopic damage score and myeloperoxidase activity (MPO) in 7.5% acetic acid (AA)-induced rectocolitis. The animals were killed 24 h after 7.5% acetic acid enema. *=P<0.05 and **= P<0.01 vs AA + saline group.

of magnitude higher than for the other tachykinin receptors (NK1 and NK3) and almost null for other nontachykinin receptors and ion channels. (Catalioto et al. 1998) This is at variance with the NK2 antagonist SR 48968 which possess measurable affinity for µ opioid receptors (Boyle et al. 1993) and local anaesthetic activity (Wang et al. 1995). For the latter antagonist the inhibitory effect afforded toward TNBS-induced ileitis in the guinea-pig was only found at a dose far from tachykinin antagonistic effect (Mazelin et al. 1998) and could be partially due to this non-tachykinin related activity. Nepadutant markedly reduced the extent of tissue injury and the associated increase in tissue permeability during the early phase of damage. Although the tachykinin NK1 receptors seems to play a major role in extravasation mechanisms, (Quartara and Maggi, 1998; Kranenveld et al. 1995; Bjorch et al. 1997) the involvement of the NK2 receptors during the inflammatory events at gastrointestinal (Mazellin et al. 1998; Lördal et al. 1996) and respiratory level (Tousignant et al. 1993) has previously described. In particular, NKA has been shown to produce a marked increase in vascular permeability at the level of lower intestine and its release can thus be part of the inflammatory response to acetic acid (Lördal et al. 1996). These results are similar to those obtained with the tachykinin NK1 receptor antagonist MEN 11467 which reduce the early but not the late phases of inflammation in the same model of rectocolitis in guinea-pigs (Cutrufo et al. 1999) or with RP 67580 and CP 96,345 in TNBSinduced colitis in rats. (Evangelista et al. 1996; Reinshagen et al. 1998; Wallace et al. 1998). However,

Neuropeptides (2000) 34(6), 355–359

TNBS provoked a decrease in rectocolonic SP and NKA levels during the acute phases of the inflammation (up to 24–48 h after ulcerogen administration) and the peptide levels were restored during the late phases (2–4 weeks later), concomitantly with an increased transcription of β-PPT mRNA in the neurons of the myenteric plexus (Renzi et al. 1994). The early tachykinin depletion seems to be one of the common events of the first phases of inflammation. The macroscopic damage induced by acetic acid was antagonized at a lesser extent by nepadutant at the top (24 h after the enema) of the acetic acid inflammation. This protective effect was not enhanced by the concomitant administration of the tachykinin NK1 receptor antagonist MEN 11467 showing a common pathway of response for the two antagonists. Furthermore in the attempt to block the development of the rectocolitis, nepadutant was given in repeated administration but the results obtained were similar to those with the single treatment indicating the involvement of tachykinins in the first phases of the inflammation and confirming a late tachykinin-independent process after acetic acid enema. (Cutrufo et al. 1999) The tissue recruitment of neutrophils was concomitant to the development of macroscopic damage and peaked at 24 h. Nepadutant markedly reduced the infiltration of neutrophils at 24 h after instillation of acetic acid. It should be noted that MPO assay is not specific for neutrophils per se, because eosinophils, monocytes and, at least in part, macrophages all possess significant hemoprotein peroxidase as well (Yamada et al. 1992). The inhibition of cell recruitment is in keeping with the known

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Effect of tachykinin NK2 receptor antagonist nepadutant in acute rectocolitis 359

effect of tachykinin NK2 receptor on inflammatory cells (e.g. influence on macrophage function see (Maggi, 1997) for review). The observation that increase in MPO activity induced by acetic acid was not prevented by the tachykinin NK1 receptor antagonist MEN 11467, supports the conclusion that the protective effects of nepadutant are indeed ascribable to selective blockade of tachykinin NK2 receptors. Taken together, the findings presented suggest the participation of tachykinin NK2 receptors in the initiation and propagation of intestinal inflammation induced by acetic acid. ACKNOWLEDGEMENTS This work was supported in part by Istituto Mobiliare Italiano (contract no. 56665). We would like to thank Dr A. Argentino-Storino for her histological work, Dr M. Tramontana for helpful advice and suggestions and Miss C. Azzurrini for her secretarial assistance. REFERENCES Bjorck S, Jennische E, Dahlstrom A, Ahlman, H (1997) Influence of topical application of drugs on dextran sulphate-induced colitis in rats. Digestive Diseases and Sciences 42: 824–832. Boyle SJ, Manley S, Tang KW et al. (1993) Affinity of the NK2 antagonist SR 48,968 at NK3 tachykinin and µ-opioid receptors. British Journal of Pharmacology 108: 24P. Catalioto R-M, Criscuoli M, Cucchi P et al. (1998) MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist. British Journal of Pharmacology 123: 81–91. Cutrufo C, Evangelista S, Cirillo R et al. (1999) Effect of MEN 11467, a new tachykinin NK1 receptor antagonist, in acute rectocolitis induced by acetic acid in guinea-pigs. European Journal of Pharmacology 374: 277–283. Evangelista S, Maggi CA, Renzetti AR (1996) Down-regulation of substance P receptors during colitis induced by trinitrobenzene sulfonic acid in rats. Neuropeptides 30: 425–428. Holzer P (1998) Implications of tachykinins and calcitonin generelated peptide in inflammatory bowel disease. Digestion 59: 269–283. Kiss S, Lecci A, de Groat WC, Maggi CA, Birder LA (1999) The effect of the NK2 receptor antagonist, MEN 11420 on proto-oncogene

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Neuropeptides (2000) 34(6), 355–359