Elsevier

Life Sciences

Volume 79, Issue 8, 17 July 2006, Pages 791-800
Life Sciences

Local anti-inflammatory effect and behavioral studies on new PDE4 inhibitors

https://doi.org/10.1016/j.lfs.2006.02.026Get rights and content

Abstract

Phosphodiesterase 4 (PDE4) inhibitors are effective anti-inflammatory drugs, although some adverse effects are observed in animals and humans. These effects have forced researchers to find new PDE4 inhibitors with less adverse effects. We recently reported the synthesis of novel heterocyclic-fused pyridazinones that inhibit PDE4. As a first step in the study of the anti-inflammatory properties of these compounds, we studied the effects of local administration of these pyridazinone derivatives in a mouse model of acute inflammation. We found that 6-Benzyl-3-methyl-4-phenylpyrazolo[3,4-d]pyridazin-7(6H)-one (CC4), ethyl 6,7-dihydro-6-ethyl-3-methyl-7-oxo-4-phenyl-thieno[2,3-d]pyridazine-2-carboxylate (CC6) and ethyl 6,7-dihydro-6-ethyl-3-methyl-4-phenyl-1H-pyrrolo[2,3-d]pyridazine-2-carboxylate (CC12) reduced the paw edema induced by zymosan in mice as rolipram (the PDE4 inhibitor prototype with anti-inflammatory activity) and indomethacin did. It is well known that rolipram locally administered induces some adverse effects such as hyperalgesia. Thus, we studied this effect after local administration of CC4, CC6 and CC12 in the formalin test. We found that CC6 induced hyperalgesic effects, whereas CC4 and CC12 did not change the nociceptive threshold. Furthermore, we found that rolipram and CC6 reduced locomotor activity, whereas CC4 and CC12 did not change locomotor performance of the mice. Since CC4 and CC12 neither affected the nociceptive threshold nor changed the locomotor performance of mice, they appear more suitable than CC6 for future studies on animals and could be developed as an anti-inflammatory drug for humans.

Introduction

Phosphodiesterases (PDEs) are enzymes responsible for the hydrolysis of the intracellular messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), causing their inactivation. cAMP and cGMP are important signalling molecules regulating inflammatory cell response. The PDEs constitute a class of at least 11 different isozymes characterized by different tissue distribution as well as by different functional roles (Huang et al., 2001).

The PDE3 family has dual specificity for both AMP and GMP cyclic nucleotides. It consists of two members, PDE3A and PDE3B, which have similar pharmacological properties but distinct expression profiles (Jeon et al., 2005). PDE3A is mainly expressed in the cardiovascular system and platelets whereas PDE3B is abundant in adipose tissue, hepatocytes, spermatocytes and in the renal collecting duct epitelium (Francis et al., 2001).

The PDE4 family of enzymes is cAMP specific and consists of four subtypes (PDE4A-PDE4D) particularly abundant in brain and immunocompetent cells as neutrophils, T-lymphocytes, macrophages and eosinophils. In these cells, PDE4 inhibitors reduce the synthesis and release of proinflammatory mediators, cytokines and active oxygen species (Jacob et al., 2002). These effects on immunocompetent cells may explain the anti-inflammatory and bronchodilatatory effects induced by PDE4 inhibitors in animal models of inflammatory diseases (Souness et al., 2000, Huang et al., 2001). In fact, much evidence has indicated the potential use of PDE4 inhibitors for the treatment of inflammatory-based disease accompanied by infiltration of tissues with inflammatory cells, such as asthma, chronic obstructive pulmonary disease and multiple sclerosis. The prototype inhibitor rolipram is the best known PDE4 inhibitor; it was clinically tested as an antidepressant several years before the discovery of its potent and selective PDE4 inhibitory activity (Wachtel, 1983, Davis, 1984). Rolipram exhibited anti-asthmatic and anti-inflammatory properties and it has shown some clinical efficacy after systemic administration, but side-effects such as nausea and emesis have prevented its development as a new antidepressant or anti-inflammatory drug (Teixeira et al., 1997). In animals, rolipram induced side-effects such as depression of locomotor activity after systemic administration (Wachtel et al., 1979, Wachtel, 1982) and markedly enhanced the hypoactivity induced by forskolin (Wachtel et al., 1987). Furthermore, rolipram enhanced the hyperalgesia induced by local administration of several pro-inflammatory agents (Cunha et al., 1999). At present, some new PDE4 inhibitors are being evaluated in the clinic such as cilomilast (Ariflo®) and roflumilast (Daxas®) but despite the anti-inflammatory effectiveness, their clinical use appears limited by the introduction of adverse effects similar to those observed after rolipram administration (Dyke and Montana, 2002).

PDE5 catalyzes the hydrolysis of cGMP, and the enzyme is abundant in lung, platelets, vascular smooth muscle and kidney. PDE5 is the primary cGMP-hydrolyzing activity in human corpus-cavernosum tissue and it is potently inhibited by sildenafil (Francis et al., 2001). The peripheral inhibition of PDE5 produces antinociception (Jain et al., 2001, Ambriz-Tututi et al., 2005) and enhances both morphine (Mixcoatl-Zecuatl et al., 2000) and diclofenac (Asomoza-Espinosa et al., 2001) antinociception in rat.

It is important to note that some other kinds of drugs, for instance, glucocorticoids, mediate important immunosuppressive and anti-inflammatory effects, but display a broad range of adverse reactions. There are continuous efforts to optimize glucocorticoid treatment. Alternative strategies such as local application or fine-tuned dose regimens have been developed over the past five decades to improve the benefit-risk ratio of these drugs (Hunter and Blyth, 1999). Alternative strategies should be developed also for PDE4 inhibitors, for instance, local application, but no data exist to our knowledge on the local anti-inflammatory effects induced by these types of drugs.

With respect to our studies on the chemistry and pharmacology of pyridazine derivatives, we recently synthesized a group of 6-aryl-4,5-heterocyclic-fused pyridazinones that demonstrated a good selectivity profile for the PDE4 family, although, to a less extent, they also can inhibit the PDE3 enzyme (Dal Piaz et al., 1997). Furthermore, it was reported that pyridazinones can potently inhibit also PDE5 enzyme (Feixas et al., 2005).

Owing to the lack of data on the anti-inflammatory effects of PDE4 inhibitors after local administration, and as a first step of the investigation on the pharmacology of these newly synthesized pyridazinones, the present study was performed in order to investigate the anti-inflammatory properties after local administration of some pyridazinones derivatives named CC4, CC6 and CC12 (Fig. 1). These were selected on the basis of their ability to inhibit PDE4 enzyme (Table 1; see also Dal Piaz et al., 1997). The anti-inflammatory activity of CC4, CC6 and CC12 was compared with rolipram and with indomethacin, a well-known anti-inflammatory drug. Since it has been reported that rolipram induces hyperalgesia after local administration (Cunha et al., 1999), the effects of the pyridazinone derivatives on nociceptive threshold were also investigated. In order to ascertain the mechanism of CC4, CC6 and CC12 action, some experiments were also performed and compared to those obtained administering 8-bromo-cAMP, a stable cAMP analogue, 8-bromo-cGMP, a stable cGMP analogue, erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA, a PDE2 inhibitor), cilostamide and cilostazol (PDE3 inhibitors) and sildenafil (PDE5 inhibitor). It is well known that rolipram reduces the spontaneous locomotor activity of animals (Wachtel et al., 1979, Wachtel, 1982) and we investigated also the effects induced by the test compounds on the locomotor activity of mice.

Section snippets

Animals

Male CD-1 mice (Charles River, Italy) weighing 25–30 g were used for all experiments. The animals were housed in colony cages (5 mice per cage) under standard light (light on from 7:00 a.m. to 7:00 p.m.), temperature (20 ± 1 °C) and relative humidity (60% ± 10%), for 1 week before the experimental sessions. Food and water were available ad libitum. Animal care and use followed the guidelines of the European Community Council (86/609/EEC).

Induction of edema in the mouse paw

Mice received a subcutaneous administration of 50 μl of zymosan 1%

Effects induced by pyridazinones derivatives on mouse paw edema

In animals treated with DMSO 30 min before zymosan, we observed an increase in paw volume that reached the maximal value 1–3 h after the injection, followed by a slight reduction of paw edema that was observed from 2 to 24 h after zymosan (Fig. 2, panels A–C). Fig. 2 also shows the time course of mouse paw edema development after the injection of CC4 (Fig. 2, panel A), C6 (Fig. 2, panel B) and CC12 (Fig. 2, panel C). Pyridazinone derivatives were able to reduce the increase in paw volume induced

Discussion

We found that two new pyridazinone derivatives, CC4 and CC12, that inhibit PDE4 and to a lesser extent PDE3, are able to induce the same anti-inflammatory effects as rolipram and indomethacin after local administration. Most importantly, CC4 and CC12, unlike rolipram, did not change the nociceptive threshold and did not change the motor activity of mice.

At present, we do not know the exact mechanism of action of CC4, CC6 and CC12, but some hypothesis can be put forward. It is well known that

Acknowledgements

This work was supported by the Italian National Institute of Health (Project no 1088/RI and 088/RI). We thank Mr. Stefano Fidanza and Mr. Adriano Urciuoli for their technical advice.

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