Involvement of I₂-imidazoline binding sites in positive and negative morphine analgesia modulatory effects

Abstract

Some studies, suggesting the involvement of I₂-imidazoline binding sites (I₂-IBS) in morphine analgesia modulation, prompted us to examine on mice antinociceptive assays the effect produced by 1 (phenyzoline), that in view of its high I₂-IBS affinity and high I₂-IBS selectivity with regard to I₁-IBS, α₂-adrenoreceptors and μ-opioid receptors might be considered the first interesting I₂-IBS ligand. The study was also applied to its ortho phenyl derivative 2 (diphenyzoline), designed and prepared in order to produce a possible modification of the biological profile of 1. Diphenyzoline (2) retains a significant I₂-IBS selectivity with regard to I₁-IBS, α₂-adrenoreceptors and μ-opioid receptors. Moreover, by the functional assays 1 and 2 proved inactive at all α₂-adrenoreceptors subtypes up to 10^− 3 M. As expected, phenyzoline and diphenyzoline, which are structurally related, highlighted an interesting “positive” or “negative”, respectively, morphine analgesia modulatory effect. In fact, 1 (s.c. 10 mg/kg) enhanced morphine analgesia (60% and 40% in mouse tail-flick and mouse hot-plate, respectively), while 2 (s.c. 10 mg/kg) decreased it (− 41% and − 20%, respectively). The ability to decrease morphine analgesia had never been observed before in I₂-IBS ligands. These effects were not affected by i.p. treatment of animals with yohimbine (a selective α₂-adrenoreceptor antagonist, 0.625 mg/kg) or efaroxan (an I₁-IBS/α₂-adrenoreceptor antagonist, 1.0 mg/kg). In contrast, they were completely reversed by i.p. treatment of animals with idazoxan (an I₂-IBS/α₂-adrenoreceptor antagonist, 2 mg/kg). Moreover, compound 2, in mouse tail-flick test, was able to potentiate by 23% the naloxone-induced decrease of morphine analgesia. Therefore, the results of this study indicate the crucial involvement of I₂-IBS in the morphine analgesia modulatory effects of 1 and 2.

Introduction

The existence of imidazoline binding sites (IBS) was hypothesized about 20 years ago (Bousquet et al., 1984). Findings from different laboratories have shown that they are widely distributed throughout the tissues of various species, including man, in both central and peripheral nervous systems. The numerous studies with radioligands and those of functional type (Molderings, 1997), apart from confirming the existence of IBS, have also revealed their heterogeneity. At present, they appear to be divided into I₁-IBS, recognized preferentially by [³H]-clonidine and related compounds and I₂-IBS by [³H]-idazoxan; still uncertain is the existence of I₃-IBS subtypes. Even though as a result of immunological studies it has been possible to ascribe the IBS nature to distinct proteins in human and rat brain, the structures of these binding proteins have not been identified yet (Dardonville and Rozas, 2004).

The I₁-IBS, located on the plasma membrane of neurons, are present at a fairly high density in the region of the medulla oblungata, that contains the sites of the hypotensive action for imidazoline-like and related drugs (Heemskerk et al., 1998). Pharmacological studies suggested that I₁-IBS are involved in the regulation of cardiovascular function (Bousquet, 2001, Molderings and Gothert, 1999), in the modulation of ocular pressure (Ogidigben and Potter, 2001), and in the secretion of renal sodium (Smyth and Penner, 1999).

The I₂-IBS, located principally in the outer membrane of mitochondria of peripheral and central tissues (Tesson and Parini, 1991, Tesson et al., 1992) are divided into I_2A- and I_2B-IBS according to their affinity for amiloride, high or low, respectively (Parini et al., 1996). Biochemical and pharmacological studies suggested a possible structural and functional correlation between I₂-IBS and monoamine oxidases (MAOs), two mitochondrial enzymes involved in the oxidate deamination of neurotransmitters (Raddatz et al., 2000, Eglen et al., 1998). I₂-IBS are involved in CNS pathologies such as Parkinson's disease (Reynolds et al., 1996), depression (Escriba et al., 1999), tolerance and addiction to opiods (Ruiz-Durantez et al., 2003). It has been described that I₂-IBS are present in brain areas involved in perception and response to painful stimuli (Ruggiero et al., 1998).

Since a potentiation of the analgesic effect of morphine by agmatine (proposed as endogenous ligand of IBS) (Kolesnikov et al., 1996) has been observed and a significant decrease of the IBS density in different brain regions after chronic morphine treatment (Su et al., 2001), it is reasonable to hypothesize IBS involvement in the modulation of pain and the pharmacological effects of opioids. However, since agmatine binds to I₂-IBS, I₁-IBS and α₂-adrenoreceptors in many regions of the brain (Li et al., 1994), it could therefore be difficult to ascribe its modulatory activity on morphine antinociception to its binding to one of these receptor types.

Recently a number of studies suggested that some substances displaying high affinity toward the I₂-IBS with regard to α₂-adrenoceptors such as BFI (Hudson et al., 1995, Alemany et al., 1997) and tracizoline (Pigini et al., 1997), named also valldemossine or LSL 61122 (Ozaita et al., 1997) were able, analogously to agmatine, to regulate opioid-induced analgesia (Sanchez-Blazquez et al., 2000), and to attenuate the development of tolerance and dependence (Boronat et al., 1998). Moreover, the I₂-IBS ligands exhibiting no antinociceptive effects by themselves but able to potentiate the analgesic effect of morphine, have been indicated as agonists, while those, such as idazoxan, that by co-treatment completely reverse this potentiation, have been considered antagonists (Sanchez-Blazquez et al., 2000).

Despite the intense efforts in the field, there is still a great deal of uncertainty about the involvement of I₂-IBS in the modulation of pain, also because the most commonly used ligands are generally scantly selective with regard to the two main subtypes I₁- and I₂-IBS, and a possible role of the I₁-IBS in analgesia cannot be ruled out (Millan, 2002).

In the present study, our lead is compound 1 (Phenyzoline) (Fig. 1), selected for its very high I₂-IBS affinity and high I₂-IBS selectivity with regard to I₁-IBS and α₂-adrenoceptors (pK_i I₂ = 8.60; pK_i I₁ = 5.43; pK_i α₂ = 5.70; I₂/I₁ = 1479; I₂/α₂ = 794) (Gentili et al., 2003). Analogously to what was demonstrated by us for ligands interacting with α₂-adrenoceptors (Gentili et al., 2002) and I₁-IBS (Gentili et al., 2005), in order to produce a possible modulation of biological profile of 1, we designed and prepared its ortho phenyl derivative, compound 2 (Diphenyzoline), already known (Jones and Dimopoulos, 2000) but never considered from this point of view (Fig. 1). The affinity values at I₂-IBS, I₁-IBS and α₂-adrenoceptors of 2 and the values at μ-opioid receptors of 1 and 2 were determined. Compounds 1 and 2 were tested on two algesiometric paradigms: mouse hot-plate and mouse tail-flick tests; the previously studied tracizoline (Pigini et al., 1997) or valldemossine (Sanchez-Blazquez et al., 2000) has been included in this study with the aim to verify the validity of our experimental conditions. To confirm the role played by I₂-IBS in the modulation of morphine analgesia, the effects of the pre-treatment with idazoxan (a mixed I₂-IBS/α₂-adrenoceptor antagonist), efaroxan (a mixed I₁-IBS/α₂-adrenoceptor antagonist), and yohimbine (a selective α₂-adrenoceptor antagonist) were evaluated; a pre-treatment with naloxone (an opioid receptor antagonist) was also performed. In addition, 1 and 2 were subjected to functional assays at α₂-adrenoceptor subtypes.

Finally, the molecular structures of 1, 2, tracizoline and idazoxan were superposed.