Terri Cummons

Species‐specific in vitro pharmacological effects of the cannabinoid receptor 2 (CB2) selective ligand AM1241 and its resolved enantiomers

Racemic (R,S) AM1241 is a cannabinoid receptor 2 (CB2)‐selective aminoalkylindole with antinociceptive efficacy in animal pain models. The purpose of our studies was to provide a characterization of R,S‐AM1241 and its resolved enantiomers in vitro and in vivo.

The persistence of a long-term negative affective state following the induction of either acute or chronic pain.

Abstract Clinically, pain is a complex phenomenon consisting of both sensory and affective aberrations that can persist indefinitely. Pre‐clinically, several animal paradigms have been established that reliably mimic both the acute and chronic aspects of pain pertinent to the human condition; however, the commonly used behavioral models only assess the sensory component of pain elicited by an evoked nociceptive stimulus. Since the affective‐motivational component of pain is an important determinant of the overall pain experience in man, we investigated how this aspect may be modeled long‐term in rats using novel objects and a modified conditioned place aversion (CPA) paradigm. Findings demonstrate that animals subjected to either neuropathic injury or inflammatory insult display a significant conditioned place aversion to a pain‐paired environment that is paralleled by an increased number of hind paw withdrawals and fewer number of novel object interactions during painful conditioning sessions. Moreover, this aversion is maintained for 1 month in the absence of further conditioning. We also determined that a non‐analgesic, non‐rewarding dose of morphine administered prior to pain‐paired conditioning sessions attenuates the pain‐induced aversion and its relative persistence in both pain models. Together, these findings underscore the importance of negative affect accompanying painful conditions and its long‐term persistence even when the injury or insult has resolved. Lastly, these results suggest how both sensory and affective aberrations associated with neuropathic‐ and inflammatory‐like conditions and the memory of such known to impact quality of life in man may be addressed pre‐clinically in rodents.

Abnormal gait, due to inflammation but not nerve injury, reflects enhanced nociception in preclinical pain models.

Validation of gait analysis has the potential to bridge the gap between data from animal pain models and clinical observations. The goal of these studies was to compare alterations in gait due to inflammation or nerve injury to traditional pain measurements in animals. Pharmacological experiments determined whether gait alterations were related to enhanced nociception, edema, or motor nerve dysfunction. Gait was analyzed using an automated system (DigiGait) after injection of an inflammatory agent (carrageenan; CARR or FCA; Freunds complete adjuvant) or nerve injury (axotomy; AXO, partial sciatic nerve ligation; PSNL, spinal nerve ligation; SNL or chronic constriction injury; CCI). All models caused significant alterations in gait and thermal (inflammatory) or mechanical (nerve injury) hyperalgesia. Both indomethacin and morphine were able to block or reverse thermal hyperalgesia and normalize gait in the CARR model. Indomethacin partially blocked and did not reverse paw edema, suggesting that gait alterations must be primarily driven by enhanced nociception. In nerve injury models, AXO, PSNL, CCI, and SNL caused changes to the largest number of gait indices with the rank order being AXO>PSNL=CCI >> SNL. Gabapentin and duloxetine reversed mechanical hyperalgesia but did not normalize gait in any nerve injury model. Collectively, these data suggest that pain is the primary driver of abnormal gait in models of inflammatory but not nerve injury-related pain and suggests that, in the latter, disruption in gait is due to perturbation to the motor system. Gait may therefore constitute an alternative and potentially clinically relevant measure of pain due to inflammation.

Pain is a salient “stressor” that is mediated by corticotropin-releasing factor-1 receptors

Corticotropin-releasing factor (CRF) plays a major role in controlling the bodys response to stress. Because painful conditions are inherently stressful, we hypothesize that CRF may act via CRF-1 receptors to contribute to the pain experience. Studies were designed to investigate whether blocking CRF-1 receptors with selective antagonists or reducing their expression with CRF-Saporin, would attenuate ulcer, inflammatory- and neuropathic-like pain. Five experimental designs were undertaken. In experiment 1, ulcer pain was induced in mice following oral administration of indomethacin, while in experiments 2 and 3, inflammatory pain was induced in rats with either carrageenan or FCA, respectively. For these studies, animals were dosed with CP-154,526 (3, 10, 30 mg/kg) and NBI 27914 (1-30 mg/kg) 1 h prior to the assessment of tactile, thermal or mechanical hypersensitivity, respectively. In experiment 4, neuropathic pain was induced. Twenty-one days following spinal nerve ligation (SNL), animals received CRF-Saporin or control. Three weeks later tactile allodynia was assessed. Similarly, in experiment 5, a separate set of rats received CRF-Saporin or control. Twenty-one days later, mechanical hyperalgesia was assessed following intraplantar carrageenan. Results from the antagonist studies showed that CP-154,526 and NBI 27914 either fully or partially reversed the referred ulcer pain with minimal effective doses (MED) equal to 3 and 10 mg/kg, respectively. Similarly, both NBI 27914 and CP-154,526 reversed the thermal and mechanical hypersensitivity elicited by carrageenan and FCA with MEDs </= 5 and 10 mg/kg, respectively. Findings from the two CRF-Saporin studies determined that pre-treatment with this toxin significantly attenuated SNL- and carrageenan-induced tactile hypersensitivity. Together, these findings suggest that CRF-1 receptors mediate pain and implicate CRF in this regard.

Pharmacological Characterization of the Muscarinic Agonist (3R,4R)-3-(3-Hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983) in in Vitro and in Vivo Models of Chronic Pain

Here, we have investigated the in vitro pharmacology of a muscarinic agonist, (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983), and we demonstrated its activity in several models of pain. WAY-132983 had a similar affinity for the five muscarinic receptors (9.4–29.0 nM); however, in calcium mobilization studies it demonstrated moderate selectivity for M1 (IC50 = 6.6 nM; Emax = 65% of 10 μM carbachol-stimulation) over the M3 (IC50 = 23 nM; Emax = 41%) and M5 receptors (IC50 = 300 nM; Emax = 18%). WAY-132983 also activated the M4 receptor, fully inhibiting forskolin-induced increase in cAMP levels (IC50 = 10.5 nM); at the M2 receptor its potency was reduced by 5-fold (IC50 = 49.8 nM). In vivo, WAY-132983 demonstrated good systemic bioavailability and high brain penetration (>20-fold over plasma levels). In addition, WAY-1329823 produced potent and efficacious antihyperalgesic and antiallodynic effects in rodent models of chemical irritant, chronic inflammatory, neuropathic, and incisional pain. It is noteworthy that efficacy in these models was observed at doses that did not produce analgesia or ataxia. Furthermore, a series of antagonist studies demonstrated that the in vivo activity of WAY-132983 is mediated through activation of muscarinic receptors primarily through the M4 receptor. The data presented herein suggest that muscarinic agonists, such as WAY-132983, may have a broad therapeutic efficacy for the treatment of pain.

Prodrugs of Perzinfotel with Improved Oral Bioavailability

Previous studies with perzinfotel (1), a potent, selective, competitive NMDA receptor antagonist, showed it to be efficacious in inflammatory and neuropathic pain models. To increase the low oral bioavailability of 1 (3-5%), prodrug derivatives (3a-h) were synthesized and evaluated. The oxymethylene-spaced diphenyl analogue 3a demonstrated good stability at acidic and neutral pH, as well as in simulated gastric fluid. In rat plasma, 3a was rapidly converted to 1 via 2a. Pharmacokinetic studies indicated that the amount of systemic exposure of 1 produced by a 10 mg/kg oral dose of 3a was 2.5-fold greater than that produced by a 30 mg/kg oral dose of 1. Consistent with these results, 3a was significantly more potent and had a longer duration of activity than 1 following oral administration in a rodent model of inflammatory pain. Taken together, these results demonstrate that an oxymethylene-spaced prodrug approach increased the bioavailability of 1.

Preclinical characterization of BRL 44408: antidepressant- and analgesic-like activity through selective α2A-adrenoceptor antagonism

Biogenic amines such as norepinephrine, dopamine, and serotonin play a well-described role in the treatment of mood disorders and some types of pain. As alpha2A-adrenoceptors regulate the release of these neurotransmitters, we examined the therapeutic potential of BRL 44408, a potent (Ki=8.5 nM) and selective (>50-fold) alpha2A-adrenoceptor antagonist (K(B)=7.9 nM). In rats, BRL 44408 penetrated the central nervous system resulting in peak brain and plasma concentrations of 586 ng/g and 1124 ng/ml, respectively. In a pharmacodynamic assay, pretreatment with BRL 44408 to rats responding under a fixed-ratio 30 operant response paradigm resulted in a rightward shift of the clonidine dose-response curve, an effect indicative of alpha2-adrenoceptor antagonism in vivo. Consistent with presynaptic autoreceptor antagonism and tonic regulation of neurotransmitter release, acute administration of BRL 44408 elevated extracellular concentrations of norepinephrine and dopamine, but not serotonin, in the medial prefrontal cortex. Additionally, BRL 44408, probably by inhibiting alpha2A heteroceptors, produced a significant increase in cortical levels of acetylcholine. In the forced swim test and schedule-induced polydipsia assay, BRL 44408 produced an antidepressant-like response by dose-dependently decreasing immobility time and adjunctive water intake, respectively, while in a model of visceral pain, BRL 44408 exhibited analgesic activity by decreasing para-phenylquinone (PPQ)-induced abdominal stretching. Finally, BRL 44408 did not produce deficits in overall motor coordination nor alter general locomotor activity. This preclinical characterization of the neurochemical and behavioural profile of BRL 44408 suggests that selective antagonism of alpha2A-adrenoceptors may represent an effective treatment strategy for mood disorders and visceral pain.

WAY-318068: a novel, potent and selective noradrenaline re-uptake inhibitor with activity in rodent models of pain and depression

Background and purpose:  Antidepressants, which raise the CNS concentrations of 5‐HT and noradrenaline, are frequently used in the treatment of chronic pain; however, it is not known if increasing CNS noradrenaline levels alone is sufficient for efficacy, in part resulting from a lack of small molecules with sufficient selectivity.