Administacion de Recursos Humanos 1

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Gabapentin (30 mg/kg; ip) and CR4056 (2 mg/kg; os) treatments significantly increased the mechanical withdrawal thresholds of both ipsilateral and contralateral hind paws compared with vehicle. No statistically significant difference was detected between groups treated with gabapentin and CR4056. The oral treatment with amitriptyline (3 mg/kg) was found to be different from vehicle treated animals only in the contralateral paw. Conversely, the oral treatment with celecoxib (30 mg/kg) failed to modulate the decrease of withdrawal thresholds induced by the acidic saline treatment both in the contralateral and in the ipsilateral paw.

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CR4056 has now completed preclinical development and a phase I safety study in humans is currently ongoing to develop the compound as a first in class imidazoline-2 ligand in chronic and neuropathic pain conditions.

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Efficacy and variability of treatments

MAO A allosteric inhibition I2-mediated

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CR4056 significantly increased the mechanical withdrawal thresholds of both ipsilateral (F [4, 30]= 18.64, P< 0.001) and contralateral (F [4, 30]= 24.13, P< 0.001) hind paws compared with vehicle. A pairwise multiple comparison procedure (HolmSidak method) further evidenced that the anti-nociceptive effects of 2 mg/kg CR4056 (orally administered) and 30 mg/kg gabapentin (i.p. administered) were similar. CR4056 (2 and 6 mg/kg) completely reversed acidic saline induced allodynia. No statistically significant difference was detected between the groups treated orally with 2 and 6 mg/kg CR4056.

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CR4056 dose-response effect:

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This study shows that low doses of CR4056 (MED= 0.6 mg/kg), a new ligand of the imidazoline-2 sites (I2R), administrated orally to rats, are effective to cure an experimental allodynia induced by repeated injections of acidic saline in the gastrocnemius muscle. Interestingly, while both Gabapentin and Amitriptyline were found to be active in this model, Celecoxib, a potent COX-2 inhibitor, failed to reverse this experimentally-induced allodynia. A previous study showed that CR4056 has a surprising efficacy in several other models of inflammatory and neuropathic pain (Ferrari et al., J Pain Res. 2011; 4:111). In all of these paradigms, oral doses of CR4056 below 20 mg/kg were highly effective against the exaggerated pain sensation, while higher doses proved to be, as in the case of opiates, decidedly hypoalgesic. However, CR4056 does not interact directly with any of the known opioid receptors. Taking into account that some imidazoline ligands are able to increase βendorphin secretion in the rat adrenal medulla (Chang et al., Neurosci Lett. 2010; 468:297), this will possibly be additive to the well described central effect induced by I2 ligands on the firing rate of locus coeruleus neurons. Moreover, CR4056 was found to selectively inhibit the activity of human recombinant MAO-A with submicromolar affinity and, when administrated orally to rats, to increase the norepinephrine tissue levels both in the cerebral cortex and in the lumbar spinal cord. Thus, a tentative picture of CR4056 mode of action could be summarized as follows:

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Results

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Acid-induced muscle allodynia in rats. Male Wistar rats (Harlan, Italy) weighing 200–300 g were used. Right gastrocnemius muscle was injected with 150µl of pH= 4 preservative-free sterile saline. Five days later, the same gastrocnemius muscle was re-injected using an identical injection protocol. As a control for the injection procedure, a separate group of animals were injected with sterile saline. Ipsilateral and contralateral paw withdrawal thresholds were measured in response to mechanical stimuli on days 0 (baseline), 5 and 24 hours after the second acid injection (day 6). Nociceptive thresholds, expressed in grams (g), were measured using a Dynamic Plantar Aesthesiometer (Ugo Basile, Italy) by applying increasing pressure to the right and left hind paw until the rat withdrew the paw. A maximal cut-off of 50g was used to prevent tissue damage. The threshold was tested three times for each paw and the mean value was calculated. CR4056 was administered as oral suspension at doses of 0.6, 2 and 6 mg/kg in comparison to gabapentin (30 mg/kg; i.p.) and/or amitriptyline (3 mg/kg; os). Mechanical withdrawal thresholds of both hind paws were measured on the sixth day (6d) from the beginning of the experiment both before and 1 hour after CR4056 and amitriptyline or 30 minutes after gabapentin injection, respectively.

Discussion & Conclusions

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Ferrari Tremolada Ugolini Lanza Caselli 1 Pharmacology & Toxicology Dept., Rottapharm|Madaus Research Center, Monza (MB), ITALY 2 Rottapharm Biotech, Basovizza (TS), ITALY

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Fibromyalgia in humans is still a medically unexplained syndrome mainly characterized by a non-inflammatory, widespread and chronic muscoskeletal pain. Thus, the efforts to develop a preclinical model of this condition should be considered as a contribution to the clinical definition of fibromyalgia. The acidic saline model of pain in rats was first described ten years ago by Sluka et al. (Muscle Nerve 2001; 24:37), and than pharmacologically characterized by the Blackburn-Munro’s group (Eur J Pharmacol 2004; 487:93). In summary, the unilateral intramuscular injection of acidic saline produces a bilateral long lasting allodynia, which is believed to be centrally mediated. This study shows the in vivo efficacy, in this experimental paradigm, of a novel imidazoline-2 ligand (2-phenyl-6-(1H-imidazol-1yl)]quinazoline; CR4056) in comparison with drugs (gabapentin and amitriptyline) belonging to the pharmacological families of the recently FDA approved drugs for fibromyalgia.

Methods

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Introduction

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Evaluation of CR4056, in comparison with Gabapentin and Amitriptyline, in a rat model of Fibromyalgia