Dual enkephalinase inhibitor PL37 as a potential novel treatment of migraine: evidence from a rat model.

The dual enkephalinase inhibitor PL37, a small molecule that protects enkephalins from rapid degradation, has demonstrated analgesic properties in animal pain models and in early human clinical trials. This study tested the antimigraine potential of PL37 on cutaneous mechanical hypersensitivity affecting cephalic regions in migraineurs. Using behavioural testing and c-Fos immunoreactivity in male rats, we investigated the effects of single (oral or intravenous) and repeated oral administration of PL37 on changes in cutaneous mechanical sensitivity and sensitization of the trigeminocervical complex induced by repeated administration of the nitric oxide donor, isosorbide dinitrate. In naïve rats, single or repeated administration of PL37 or vehicle had no effect on cephalic mechanical sensitivity. However, single oral PL37 treatment effectively inhibited isosorbide dinitrate-induced acute cephalic mechanical hypersensitivity. Single intravenous but not oral PL37 administration inhibited chronic cephalic mechanical hypersensitivity. Daily oral administration of PL37 prevented cephalic mechanical hypersensitivity and decreased touch-induced c-Fos expression in trigeminocervical complex following repeated isosorbide dinitrate administration. These data reveal the therapeutic potential of the dual enkephalinase inhibitor PL37 as an acute and prophylactic treatment for migraine. Protecting enkephalins from their degrading enzymes therefore appears to be an innovative approach to treat migraine.

Chronic facial inflammatory pain-induced anxiety is associated with bilateral deactivation of the rostral anterior cingulate cortex.

Patients with chronic pain, especially orofacial pain, often suffer from affective disorders, including anxiety. Previous studies largely focused on the role of the caudal anterior cingulate cortex (cACC) in affective responses to pain, long-term potentiation (LTP) in cACC being thought to mediate the interaction between anxiety and chronic pain. But recent evidence indicates that the rostral ACC (rACC), too, is implicated in processing affective pain. However, whether such processing is associated with neuronal and/or synaptic plasticity is still unknown. We addressed this issue in a chronic facial inflammatory pain model (complete Freund's adjuvant model) in rats, by combining behavior, Fos protein immunochemistry and ex vivo intracellular recordings in rACC slices prepared from these animals. Facial mechanical allodynia occurs immediately after CFA injection, peaks at post-injection day 3 and progressively recovers until post-injection days 10-11, whereas anxiety is delayed, being present at post-injection day 10, when sensory hypersensitivity is relieved, but, notably, not at post-injection day 3. Fos expression reveals that neuronal activity follows a bi-phasic time course in bilateral rACC: first enhanced at post-injection day 3, it gets strongly depressed at post-injection day 10. Ex vivo recordings from lamina V pyramidal neurons, the rACC projecting neurons, show that both their intrinsic excitability and excitatory synaptic inputs have undergone long-term depression (LTD) at post-injection day 10. Thus chronic pain processing is associated with dynamic changes in rACC activity: first enhanced and subsequently decreased, at the time of anxiety-like behavior. Chronic pain-induced anxiety might thus result from a rACC deactivation-cACC hyperactivation interplay.

Postnatal development of inner lamina II interneurons of the rat medullary dorsal horn.

ABSTRACT: Pain processing in young mammals is immature. Despite the central role of the medullary dorsal horn (MDH) in processing orofacial sensory information, the maturation of the neurons within the MDH has been largely overlooked. Combining in vitro electrophysiological recordings and 3D morphological analysis over the first postnatal month in rats, we investigated the age-dependent development of the neurons within the inner lamina II (IIi) of the MDH. We show the lamina IIi neuronal population transition into a more hyperpolarized state, with modification of the action potential waveform, and a shift from single spiking, at early postnatal ages, to tonic firing and initial bursting at later stages. These physiological changes are associated with a strong structural remodelling of the neuronal morphology with most of the modifications occurring after the third postnatal week. Among the lamina IIi neuronal population, the subpopulation of interneurons expressing the γ isoform of the protein kinase C (PKCγ+) are key elements for the circuits underlying facial mechanical allodynia. How do they develop from the rest of the lamina IIi constitute an important question that remained to be addressed. Here, we show that PKCγ+ interneurons display electrophysiological changes over time comparable with the PKCγ- population. However, they show a distinctive increase of the soma volume and primary branches length, as opposed to the PKCγ- population. Together, our data demonstrate a novel pattern of late postnatal maturation of lamina IIi interneurons, with a spotlight on PKCγ+ interneurons, that may be relevant for the development of orofacial sensitivity.

Improved potency of pyridin-2(1H)one derivatives for the treatment of mechanical allodynia.

Mechanical allodynia, a painful sensation caused by innocuous touch, is a major chronic pain symptom, which often remains without an effective treatment. There is thus a need for new anti-allodynic treatments based on new drug classes. We recently synthetized new 3,5-disubstituted pyridin-2(1H)-one derivatives. By substituting the pyridinone at the 3-position by various aryl/heteroaryl moieties and at the 5-position by a phenylamino group, we discovered that some derivatives exhibited a strong anti-allodynic potency in rats. Here, we report that varying the substitution of the pyridinone 5-position, the 3-position being substituted by an indol-4-yl moiety, further improves such anti-allodynic potency. Compared with 2, one of the two most active compounds of the first series, eleven out of nineteen newly synthetized compounds showed higher anti-allodynic potency, with two of them completely preventing mechanical allodynia. In the first series, hit compounds 1 and 2 appeared to be inhibitors of p38α MAPK, a protein kinase known to underlie pain hypersensitivity in animal models. Depending on the substitution at the 5-position, some newly synthetized compounds were also stronger p38α MAPK inhibitors. Surprisingly, though, anti-allodynic effects and p38α MAPK inhibitory potencies were not correlated, suggesting that other biological target(s) is/are involved in the analgesic activity in this series. Altogether, these results confirm that 3,5-disubstituted pyridine-2(1H)-one derivatives are of high interest for the development of new treatment of mechanical allodynia.

Pyridin-2(1H)one derivatives: A possible new class of therapeutics for mechanical allodynia.

Mechanical Allodynia (MA), a frequent chronic pain symptom caused by innocuous stimuli, constitutes an unmet medical need, as treatments using analgesics available today are not always effective and can be associated with important side-effects. A series of 3,5-disubstituted pyridin-2(1H)-ones was designed, synthesized and evaluated in vivo toward a rat model of inflammatory MA. We found that the series rapidly and strongly prevented the development of MA. 3-(2-Bromophenyl)-5-(phenylamino)pyridin-2(1H)-one 69, the most active compound of the series, was also able to quickly reverse neuropathic MA in rats. Next, when 69 was evaluated toward a panel of 50 protein kinases (PK) in order to identify its potential biological target(s), we found that 69 is a p38α MAPK inhibitor, a PK known to contribute to pain hypersensitivity in animal models. 3,5-Disubstituted pyridin-2(1H)-ones thus could represent a novel class of analgesic for the treatment of MA.

Recurrent administration of the nitric oxide donor, isosorbide dinitrate, induces a persistent cephalic cutaneous hypersensitivity: A model for migraine progression.

Background A subgroup of migraineurs experience an increase in attack frequency leading to chronic migraine. Methods We assessed in rats the roles of dose and repeat administration of systemic isosorbide dinitrate (ISDN), a nitric oxide donor, on the occurrence and development of cephalic/face and extracephalic/hindpaw mechanical allodynia as a surrogate of migraine pain, and the effect of acute systemic sumatriptan and olcegepant and chronic systemic propranolol on these behavioral changes. Results A single high (H-ISDN) but not low (L-ISDN) dose of ISDN induces a reversible cephalic and extracephalic mechanical allodynia. However, with repeat administration, L-ISDN produces reversible cephalic but never extracephalic allodynia, whereas H-ISDN induces cephalic and extracephalic allodynia that are both potentiated. H-ISDN-induced cephalic allodynia thus gains persistency. Sumatriptan and olcegepant block single H-ISDN-induced behavioral changes, but only olcegepant reduces these acute changes when potentiated by repeat administration. Neither sumatriptan nor olcegepant prevent chronic cephalic hypersensitivity. Conversely, propranolol blocks repeat H-ISDN-induced chronic, but not acute, behavioral changes. Conclusions Repeated ISDN administration appears to be a naturalistic rat model for migraine progression, suitable for screening acute and preventive migraine therapies. It suggests frequent and severe migraine attacks associated with allodynia may be a risk factor for disease progression.

The nitric oxide donor, isosorbide dinitrate, induces a cephalic cutaneous hypersensitivity, associated with sensitization of the medullary dorsal horn.

Nitric oxide donors are known to produce headache in healthy as well as migraine subjects, and to induce extracephalic cutaneous hypersensitivity in rodents. However, little is known on the effect of nitric oxide donors on cephalic cutaneous sensitivity. Combining behavioral, immunohistochemical, and in vivo electrophysiological approaches, this study investigated the effect of systemic administration of the nitric oxide donor, isosorbide dinitrate (ISDN), on cephalic and extracephalic cutaneous sensitivity and on neuronal activation within the medullary dorsal horn (MDH) in the rat. Systemic administration of ISDN increased selectively the first phase and interphase of the facial formalin test, but had no effect on the hindpaw formalin one. Monitoring neuronal activity within the MDH with phospho-ERK1/2 immunoreactivity revealed that ISDN alone did not activate MDH neurons, but significantly increased the number of formalin-evoked phospho-ERK1/2-immunoreactive cells in the ipsilateral, but not contralateral, MDH. Using in vivo electrophysiological unit recordings, we show that ISDN administration never affected the spontaneous activity of trigeminal wide dynamic range neurons, but, facilitated C-fiber-evoked responses in half the neurons tested. This research demonstrates that a nitric oxide donor, isosorbide dinitrate, induces selectively cephalic hyperalgesia that arises as a consequence of central sensitization in pain pathways that subserve meningeal nociception. This model better mimics the clinical condition and offers another possibility of studying the role of nitric oxide donor in the physiopathology of headache.

Effects of glia metabolism inhibition on nociceptive behavioral testing in rats.

Fluoroacetate has been widely used to inhibit glia metabolism in vivo. It has yet to be shown what the effects of chronic intrathecal infusion of fluoroacetate on nociceptive behavioral testing are. The effects of chronic infusion of fluoroacetate (5 nmoles/h) for 2 weeks were examined in normal rats. Chronic intrathecal fluoroacetate did not alter mechanical threshold (von Frey filaments), responses to supra-threshold mechanical stimuli (von Frey filaments), responses to hot (hot plate) or cool (acetone test) stimuli and did not affect motor performance of the animals, which was tested with rotarod. This suggests that fluoroacetate at appropriate dose did not suppress neuronal activity in the spinal cord.

Activation of medullary dorsal horn γ isoform of protein kinase C interneurons is essential to the development of both static and dynamic facial mechanical allodynia.

The γ isoform of protein kinase C (PKCγ), which is concentrated in a specific class of interneurons within inner lamina II (IIi ) of the spinal dorsal horn and medullary dorsal horn (MDH), is known to be involved in the development of mechanical allodynia, a widespread and intractable symptom of inflammatory or neuropathic pain. However, although genetic and pharmacological impairment of PKCγ were shown to prevent mechanical allodynia in animal models of pain, after nerve injury or reduced inhibition, the functional consequences of PKCγ activation alone on mechanical sensitivity are still unknown. Using behavioural and anatomical approaches in the rat MDH, we tested whether PKCγ activation in naive animals is sufficient for the establishment of mechanical allodynia. Intracisternal injection of the phorbol ester, 12,13-dibutyrate concomitantly induced static as well as dynamic facial mechanical allodynia. Monitoring neuronal activity within the MDH with phospho-extracellular signal-regulated kinases 1 and 2 immunoreactivity revealed that activation of both lamina I-outer lamina II and IIi -outer lamina III neurons, including lamina IIi PKCγ-expressing interneurons, was associated with the manifestation of mechanical allodynia. Phorbol ester, 12,13-dibutyrate-induced mechanical allodynia and associated neuronal activations were all prevented by inhibiting selectively segmental PKCγ with KIG31-1. Our findings suggest that PKCγ activation, without any other experimental manipulation, is sufficient for the development of static and dynamic mechanical allodynia. Lamina IIi PKCγ interneurons have been shown to be directly activated by low-threshold mechanical inputs carried by myelinated afferents. Thus, the level of PKCγ activation within PKCγ interneurons might gate the transmission of innocuous mechanical inputs to lamina I, nociceptive output neurons, thus turning touch into pain.

Neuropathic pain depends upon D-serine co-activation of spinal NMDA receptors in rats.

Activation of N-methyl-d-aspartate (NMDA) receptors is critical for hypersensitivity in chronic neuropathic pain. Since astroglia can regulate NMDA receptor activation by releasing the NMDA receptor co-agonist d-serine, we investigated the role of NMDA receptor and d-serine in neuropathic chronic pain. Male Wistar rats underwent right L5-L6 spinal nerve ligation or sham surgery and were tested for mechanical allodynia and hyperalgesia after 14 days. Acute intrathecal administration of the NMDA receptor antagonist d-AP5 as well as chronic administration of the glia metabolism inhibitor fluoroacetate significantly reduced mechanical allodynia in neuropathic rats. The effect of fluoroacetate was reversed by acutely administered intrathecal d-serine. Degrading d-serine using acute intrathecal administration of d-aminoacid oxidase also reduced pain symptoms. Immunocytochemistry showed that about 70% of serine racemase, the synthesizing enzyme of d-serine, was expressed in astrocyte processes in the superficial laminae of L5 dorsal horn. Serine racemase expression was upregulated in astrocyte processes in neuropathic rats compared to sham rats. These results show that neuropathic pain depends upon glial d-serine that co-activates spinal NMDA receptors.