Anxiolytic-like effects of mirogabalin, a novel ligand for α2δ ligand of voltage-gated calcium channels, in rats repeatedly injected with acidic saline intramuscularly, as an experimental model of fibromyalgia.

BACKGROUND: Mental disorders including anxiety and depression are common comorbidities in fibromyalgia patients, and exert a profound impact on their quality of life. Mirogabalin, a novel ligand for the α2δ-subunit of voltage-gated calcium channels, shows analgesic effects in fibromyalgia and neuropathic pain models. To provide additional information regarding its potential utility for treating chronic pain, we examined its anxiolytic-like effects in rats repeatedly injected with acidic saline intramuscularly (Sluka model), as an experimental fibromyalgia model. METHODS: Male Sprague-Dawley rats received two intramuscular injections of acidic saline (pH 4.0) into the gastrocnemius muscle. After the development of tactile allodynia demonstrated by decreased paw withdrawal threshold to von Frey filaments, anxiety-like behaviours were evaluated using the open field test and the elevated plus maze test. RESULTS: Sluka model rats exhibited anxiety-like behaviours in the open field test (significant decreases in distance travelled and time spent in the central area, and significant increases in time spent in the wall area) and the elevated plus maze test (significant decreases in time spent in the open arms and significant increases in time spent in the closed arms). A single oral dose of mirogabalin (3 or 10 mg/kg) significantly alleviated and normalised these anxiety-like behaviours. CONCLUSIONS: Sluka model rats exhibited anxiety-like behaviours in the open field test and the elevated plus maze test, but mirogabalin alleviated these behaviours. Mirogabalin might thus have the potential to relieve anxiety in fibromyalgia patients.

Analgesic effects of mirogabalin, a novel ligand for α2δ subunit of voltage-gated calcium channels, in experimental animal models of fibromyalgia.

Mirogabalin, a novel ligand for the α2δ subunit of voltage-gated calcium channels, is under the development for the treatment of neuropathic pain. Mirogabalin specifically and potently binds to α2δ subunits, and it shows analgesic effects in both peripheral and central neuropathic pain models in rats. To expand pharmacological findings on mirogabalin and provide additional information of its potential for chronic pain therapy, we examined the effects of mirogabalin in 2 experimental models of fibromyalgia, namely, the intermittent cold stress model (ICS model) and the unilateral intramuscular acidic saline injection model (Sluka model). To induce chronic mechanical hypersensitivity, mice were placed under ICS conditions for 3 days, whereas rats were injected twice with acidic saline (pH 4) into the gastrocnemius muscle in a 4-day interval. The pain sensitivity was evaluated by the von Frey test. Long-lasting increases in pain response score or decreases in pain threshold to the von Frey stimulation were observed in both the ICS and Sluka models. Mirogabalin (1, 3, or 10 mg/kg, p.o.) dose-dependently alleviated the mechanical hypersensitivity, with significant effects persisting at 6 or 8 h following administration. The standard α2δ ligand, pregabalin (30 mg/kg, p.o.), also significantly reduced the mechanical hypersensitivity. In summary, mirogabalin showed analgesic effects in the ICS model mice and in the Sluka model rats. Therefore, mirogabalin may have the potential to provide effective pain relief in patients with fibromyalgia.

Alkylsulfanyl analogs as potent α2δ ligands.

We identified novel (3R, 5S)-3-aminomethyl-5-methanesulfanyl hexanoic acid (5a: DS75091588) and (3R, 5S)-3-aminomethyl-5-ethanesulfanyl hexanoic acid (6a: DS18430756) as sulfur-containing γ-amino acid derivatives that were useful for the treatment of neuropathic pain. These two compounds exhibited a potent analgesic effect in animal models of both type I diabetes and type II diabetes, and good pharmacokinetics.

Binding Characteristics and Analgesic Effects of Mirogabalin, a Novel Ligand for the α2δ Subunit of Voltage-Gated Calcium Channels.

Mirogabalin ([(1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl]acetic acid), a novel ligand for the α2δ subunit of voltage-gated calcium channels, is being developed to treat pain associated with diabetic peripheral neuropathy and postherpetic neuralgia. In the present study, we investigated the in vitro binding characteristics and in vivo analgesic effects of mirogabalin compared with those of pregabalin, a standard α2δ ligand. Mirogabalin showed potent and selective binding affinities for the α2δ subunits, while having no effects on 186 off-target proteins. Similar to pregabalin, mirogabalin did not show clear subtype selectivity (α2δ-1 vs. α2δ-2) or species differences (human vs. rat). However, in contrast to pregabalin, mirogabalin showed greater binding affinities for human α2δ-1, human α2δ-2, rat α2δ-1, and rat α2δ-2 subunits; further, it had a slower dissociation rate for the α2δ-1 subunit than the α2δ-2 subunit. Additionally, in experimental neuropathic pain models, partial sciatic nerve ligation rats and streptozotocin-induced diabetic rats, mirogabalin showed more potent and longer lasting analgesic effects. In safety pharmacological evaluations, mirogabalin and pregabalin inhibited rota-rod performance and locomotor activity in rats; however, the safety indices of mirogabalin were superior to those of pregabalin. In conclusion, mirogabalin shows potent and selective binding affinities for the human and rat α2δ subunits, and slower dissociation rates for the α2δ-1 subunit than the α2δ-2 subunit. It shows potent and long-lasting analgesic effects in rat models of neuropathic pain, and wider safety margins for side effects of the central nervous system. These properties of mirogabalin can be associated with its unique binding characteristics.

Endogenous opioid activity in the anterior cingulate cortex is required for relief of pain.

Pain is aversive, and its relief elicits reward mediated by dopaminergic signaling in the nucleus accumbens (NAc), a part of the mesolimbic reward motivation pathway. How the reward pathway is engaged by pain-relieving treatments is not known. Endogenous opioid signaling in the anterior cingulate cortex (ACC), an area encoding pain aversiveness, contributes to pain modulation. We examined whether endogenous ACC opioid neurotransmission is required for relief of pain and subsequent downstream activation of NAc dopamine signaling. Conditioned place preference (CPP) and in vivo microdialysis were used to assess negative reinforcement and NAc dopaminergic transmission. In rats with postsurgical or neuropathic pain, blockade of opioid signaling in the rostral ACC (rACC) inhibited CPP and NAc dopamine release resulting from non-opioid pain-relieving treatments, including peripheral nerve block or spinal clonidine, an α2-adrenergic agonist. Conversely, pharmacological activation of rACC opioid receptors of injured, but not pain-free, animals was sufficient to stimulate dopamine release in the NAc and produce CPP. In neuropathic, but not sham-operated, rats, systemic doses of morphine that did not affect withdrawal thresholds elicited CPP and NAc dopamine release, effects that were prevented by blockade of ACC opioid receptors. The data provide a neural explanation for the preferential effects of opioids on pain affect and demonstrate that engagement of NAc dopaminergic transmission by non-opioid pain-relieving treatments depends on upstream ACC opioid circuits. Endogenous opioid signaling in the ACC appears to be both necessary and sufficient for relief of pain aversiveness.

Olmesartan medoxomil ameliorates sciatic nerve regeneration in diabetic rats.

To evaluate the effect of angiotensin II type1 receptor blocker on nerve regeneration delay in diabetic rats, nerve regeneration was monitored by a pinch test on the crushed sciatic nerves of streptozotocin-induced diabetic rats. Nerve regeneration was significantly delayed in diabetic rats and was partly ameliorated by treatment with olmesartan medoxomil (3 mg/kg/day, orally). In the ipsilateral dorsal root ganglia, the mRNA level of insulin-like growth factor-1 and ciliary neurotrophic factor (CNTF) was downregulated, whereas the mRNA level of neurotrophin-3 and CNTF receptor was upregulated. Olmesartan medoxomil significantly enhanced the CNTF expression. These results showed that angiotensin II type1 receptor blocker treatment is effective on nerve regeneration delay in diabetic animals and may provide an effective therapy for clinical diabetic neuropathy.

Role of angiotensin II type 1 receptor on retinal vascular leakage in a rat oxygen-induced retinopathy model.

AIM: To investigate the role of angiotensin type 1 (AT1) and type 2 (AT2) receptors in hypoxia-induced retinal vascular hyperpermeability. METHODS: Brown-Norway rat pups were exposed to hyperoxic conditions from postnatal day 7 (P7) to P12, and to subsequent normal air for 5 days [oxygen-induced retinopathy (OIR) model]. Olmesartan medoxomil (AT1 receptor antagonist; administered orally), PD123319 (AT2 receptor antagonist; administered subcutaneously) or a vehicle was administered once daily during the last 5 days. At P16, the retinal permeability was determined by measuring the leaked fluorescein-conjugated dextran concentration in the retina. The vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) alpha proteins in the retina were assessed by an ELISA and western blotting, respectively. RESULTS: Olmesartan medoxomil partially, but significantly, inhibited the retinal vascular hyperpermeability induced by hypoxia. In contrast, PD123319 did not show a significant effect. The VEGF and HIF-1alpha protein levels were significantly elevated in the OIR retina; however, there was no significant effect of olmesartan medoxomil on the expression of either protein. CONCLUSIONS: These results suggest that the AT1 receptor is, at least partly, responsible for hyperpermeability in the OIR rat retina via a mechanism independent of HIF-1 and VEGF expression.

Inhibition of self-renewal and induction of neural differentiation by PACAP in neural progenitor cells.

Several lines of evidence have suggested roles for pituitary adenylate cyclase-activating polypeptide (PACAP) in the developing nervous system. Previously, we showed that mRNA for PACAP, vasoactive intestinal peptide (VIP), and their three receptor subtypes, is differentially expressed in embryonic stem (ES) cells, ES cell-derived, neural stem cell-enriched cultures, and differentiated neurons, by using the five steps of the in vitro neuronal culture model of ES cell differentiation. Here, we examined the effects of PACAP on self-renewal and cell lineage determination of neural progenitor/stem cells. PACAP inhibited the basic fibroblast growth factor-induced proliferation (self-renewal), as assessed by neurosphere formation. PACAP increased microtubule-associated protein 2-positive neurons without affecting the number of cells positive for the neural stem cell marker nestin, astrocyte marker glial fibrillary acidic protein, and oligodendrocyte marker CNPase. These results suggest that PACAP inhibits self-renewal but, instead, induces early neuronal differentiation of neural progenitor cells.

Pharmacological and pharmacokinetic study of olmesartan medoxomil in animal diabetic retinopathy models.

A close relationship between the renin-angiotensin system and the pathophysiology of diabetic retinopathy has been suggested, several angiotensin II type 1 receptor (angiotensin AT1 receptor) antagonists being effective in animal models. Therefore, we examined the efficacy of an angiotensin AT1 receptor antagonist, olmesartan medoxomil (CS-866), in animal retinopathy models. In diabetic stroke-prone spontaneously hypertensive (SHRSP) rats, 4-week treatment with CS-866 prevented the elongation of oscillatory potential peaks dose-dependently which almost normalized at 3 mg/kg/day. Next, in oxygen-induced retinopathy mice, CS-866 at 1 mg/kg significantly prevented the retinal neovascularization. In these animal models, plasma concentrations of CS-866 were comparable to the in vitro IC50 value of the angiotensin AT1 receptor. In summary, our data demonstrated that CS-866 was effective in early and late stage retinopathy models through the inhibition of the angiotensin AT1 receptor. These findings suggest the possibility of CS-866 as a therapeutic agent for diabetic retinopathy.

Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells.

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.

Possible involvement of a cyclic AMP-dependent mechanism in PACAP-induced proliferation and ERK activation in astrocytes.

In cultured astrocytes, PACAP activates extracellular signal-regulated kinase (ERK) and induces cell proliferation at picomolar concentrations. Here, we examined the role of cyclic AMP signaling underlying the effects of PACAP. PACAP38 induced accumulation of cyclic AMP in astrocytes at concentrations as low as 10(-12)M. PACAP38 (10(-12)-10(-9)M)-stimulated cell proliferation was completely abolished by the cyclic AMP antagonist Rp-cAMP, whereas the protein kinase A (PKA) inhibitor H89 had no effect. This PACAP38-mediated effect was also abolished by the ERK kinase inhibitor PD98059, suggesting the involvement of ERK in PACAP-induced proliferation. PACAP38 (10(-12)M)-stimulated phosphorylation of ERK lasted for at least 60 min. This effect was completely abolished by Rp-cAMP but not by H89. Dibutyryl cyclic AMP maximally stimulated the incorporation of thymidine and activation of ERK at 10(-10)M. These results suggest that PACAP-mediated stimulation of ERK activity and proliferation of astrocytes may involve a cyclic AMP-dependent, but PKA-independent, pathway.