Discovery of clinical candidate Sivopixant (S-600918): Lead optimization of dioxotriazine derivatives as selective P2X3 receptor antagonists.

In previous work, we discovered a lead compound and conducted initial SAR studies on a novel series of dioxotriazines to identify the compound as one of the P2X3 receptor antagonists. This compound showed high P2X3 receptor selectivity and a strong analgesic effect. Although not selected for clinical development, the compound was evaluated from various aspects as a tool compound. In the course of the following study, the molecular structures of the dioxotriazines were modified based on pharmacokinetic/pharmacodynamic (PK/PD) analyses. As a result of these SAR studies, Sivopixant (S-600918) was identified as a clinical candidate with potent and selective antagonistic activity (P2X3 IC50, 4.2 nM; P2X2/3 IC50, 1100 nM) and a strong analgesic effect in the rat partial sciatic nerve ligation model (Seltzer model) of allodynia (ED50, 0.4 mg/kg).

Dorsal horn interneuron-derived Netrin-4 contributes to spinal sensitization in chronic pain via Unc5B.

Because of the incomplete understanding of the molecular mechanisms that underlie chronic pain, the currently available treatments for this type of pain remain inefficient. In this study, we show that Netrin-4, a member of the axon guidance molecule family, was expressed in dorsal horn inner lamina II excitatory interneurons in the rat spinal cord. A similar expression pattern for Netrin-4 was also observed in human spinal cord. Behavioral analysis revealed that tactile and heat hyperalgesia after peripheral nerve injury or inflammation were abolished in Netrin-4-mutant rats. Transient suppression of Netrin-4 or its receptor Unc5B after injury could also prevent allodynia. Conversely, intrathecal administration of Netrin-4 protein to naive rats enhanced excitatory synaptic transmission in the dorsal horn and induced allodynia, suggesting that Netrin-4 is involved in spinal sensitization. Furthermore, the Unc5B receptor and subsequent activation of the tyrosine phosphatase SHP2 mediated Netrin-4-induced pain signaling in the spinal cord. These results identify Netrin-4 as a novel protein regulating spinal sensitization leading to chronic pain. Our findings provide evidence for the function of Netrin in the adult nervous system, as well as a previously unknown function in inducing pain signals from dorsal horn interneurons.

Injury-specific functional alteration of N-type voltage-gated calcium channels in synaptic transmission of primary afferent C-fibers in the rat spinal superficial dorsal horn.

We investigated functional alterations of voltage-gated calcium channels (VGCCs) in excitatory synaptic transmission from primary afferent A- and C-fibers after peripheral nerve injury. Patch-clamp recordings were performed on substantia gelatinosa (SG) neurons of spinal cord slices with an attached dorsal root, prepared from L5 spinal nerve-ligated (SNL) rats. The effects of neuronal VGCC blockers, ω-conotoxin GVIA (ω-CgTX) for N-type channels and ω-agatoxin IVA (ω-AgaIVA) for P/Q-type channels, on evoked excitatory postsynaptic currents (eEPSCs) by stimulation of A- or C-fibers were studied. Besides, electrophysiological assay using dorsal root ganglion (DRG) and immunohistochemistry were done. In naïve rats, ω-CgTX (0.1-1µM) reduced more effectively A-fiber eEPSCs than C-fiber ones. After nerve injury, ω-CgTX produced great inhibition of C-fiber eEPSCs in slices with the injured L5 dorsal root of SNL model rats, as compared to sham-operated rats. By contrast, in slices with the non-injured L4 one, inhibitory effects of ω-CgTX were not changed. This occurred concurrently with increased expression of N-type VGCCs in L5 spinal dorsal horn and with enhanced Ca(2+) currents through N-type VGCCs in small-sized (C-type) L5 DRG. In terms of A-fiber eEPSCs, ω-CgTX elicited similar inhibition in nerve-injured and sham-operated rats. ω-AgaIVA (0.1µM) had less effect on A- or C-fiber eEPSCs. These results indicate that N-type, but not P/Q-type, VGCCs mainly contribute to excitatory synaptic transmission from A- and C-fibers in the spinal dorsal horn. More importantly, following nerve injury, the functional contribution of N-type VGCCs to nociceptive transmission is increased in the pre-synaptic terminals of injured C-fibers.

Effect of the norepinephrine transporter (NET) inhibition on µ-opioid receptor (MOR)-induced anti-nociception in a bone cancer pain model.

Although norepinephrine transporter (NET) inhibition has an additional effect on µ-opioid receptor (MOR)-mediated anti-nociception in inflammatory and neuropathic pain, its effect on cancer pain is not well characterized. We investigated the additional effect of NET inhibition on MOR activation using a mouse femur bone cancer (FBC) pain model by comparing the anti-nociceptive effect of the dual-acting opioids tramadol and tapentadol and the clinically used MOR-targeted opioids oxycodone and morphine. The anti-nociceptive effects of subcutaneously administered opioids were assessed using the von-Frey filament test. Oxycodone (1 - 10 mg/kg) and morphine (5 - 50 mg/kg) dose-dependently exhibited potent anti-nociceptive effects, whereas tramadol (10 - 56 mg/kg) and tapentadol (10 - 30 mg/kg) exhibited partial effects. Rota-rod analyses of tapentadol at a higher dose (> 30 mg/kg) showed a significant decrease in motor coordination, which was partially recovered by pretreatment with MOR or α(1)-adrenoceptor antagonists. The partial anti-nociceptive effect of tapentadol (30 mg/kg) was completely suppressed by a MOR antagonist, but not by α(1)- or α(2)-adrenoceptor antagonists, suggesting that neither α(1)-adrenoceptor- nor α(2)-adrenoceptor-mediated pathways are involved in anti-nociception in the FBC model. We conclude that addition of NET inhibition does not contribute to MOR-mediated anti-nociception in bone cancer pain.

Differential activation of the µ-opioid receptor by oxycodone and morphine in pain-related brain regions in a bone cancer pain model.

BACKGROUND AND PURPOSE: Bone cancer pain is chronic and often difficult to control with opioids. However, recent studies have shown that several opioids have distinct analgesic profiles in chronic pain. EXPERIMENTAL APPROACH: To clarify the mechanisms underlying these distinct analgesic profiles, functional changes in the µ-opioid receptor were examined using a mouse femur bone cancer (FBC) model. KEY RESULTS: In the FBC model, the B(max) of [(3) H]-DAMGO binding was reduced by 15-45% in the periaqueductal grey matter (PAG), region ventral to the PAG (vPAG), mediodorsal thalamus (mTH), ventral thalamus and spinal cord. Oxycodone (10(-8) -10(-5) M) and morphine (10(-8) -10(-5) M) activated [(35) S]-GTPγS binding, but the activation was significantly attenuated in the PAG, vPAG, mTH and spinal cord in the FBC model. Interestingly, the attenuation of oxycodone-induced [(35) S]-GTPγS binding was quite limited (9-26%) in comparison with that of morphine (46-65%) in the PAG, vPAG and mTH, but not in the spinal cord. Furthermore, i.c.v. oxycodone at doses of 0.02-1.0 µg per mouse clearly inhibited pain-related behaviours, such as guarding, limb-use abnormalities and allodynia-like behaviour in the FBC model mice, while i.c.v. morphine (0.05-2.0 µg per mouse) had only partial or little analgesic effect on limb-use abnormalities and allodynia-like behaviour. CONCLUSION AND IMPLICATIONS: These results show that µ-opioid receptor functions are attenuated in several pain-related regions in bone cancer in an agonist-dependent manner, and suggest that modification of the µ-opioid receptor is responsible for the distinct analgesic effect of oxycodone and morphine.

Distinct relations among plasma concentrations required for different pharmacological effects in oxycodone, morphine, and fentanyl.

Several clinical reports showed that adverse effect profiles are not the same in morphine, oxycodone, and fentanyl. The authors investigated whether the relationship between plasma concentrations for antinociceptive effect and for various pharmacological effects differed among oxycodone, morphine, and fentanyl under controlled experimental setting using animal models. Oxycodone induced constipation and an antinociceptive effect in a similar concentration-dependent manner, whereas morphine required approximately 9-fold higher plasma concentration for antinociceptive effect compared with that for constipation when 50% effective plasma concentration (EC(50)) levels were compared. The EC(50) values for inhibition of behavioral activity were 2.1-, 2.7-, and 1.3-fold higher than those for antinociceptive effect in oxycodone, morphine, and fentanyl, respectively. Respiratory inhibition was observed even at higher plasma concentrations in all three opioids, and the differences in the EC(50) values compared with those for antinociceptive effects were 234.5-fold (oxycodone), 233.1-fold (morphine), or 104.2-fold (fentanyl). These results showed that oxycodone, morphine, and fentanyl exhibited unique patterns of plasma concentrations required for different pharmacological effects. The different adverse effect profiles observed in a clinical setting appear to be resulted from, at least in part, distinct intrinsic pharmacological profiles among these µ-opioid receptor agonists.

Morphine, oxycodone, and fentanyl exhibit different analgesic profiles in mouse pain models.

Morphine, oxycodone, and fentanyl are clinically prescribed drugs for the management of severe pain. We investigated whether these opioids possess different efficacy profiles on several types of pain in mouse pain models. When the three opioids were tested in the femur bone cancer model, all of them significantly reversed guarding behavior, whereas the effects on limb-use abnormality and allodynia-like behavior differed among the opioids. Particularly, although oxycodone (5 - 20 mg/kg) and fentanyl (0.2 mg/kg) significantly reversed limb-use abnormality, not even a high dose of morphine (50 mg/kg) could reverse it. When the effects of these opioids were examined in a sciatic nerve ligation (SNL) model of neuropathic pain, oxycodone was the most effective, producing an antinociceptive effect without affecting the withdrawal threshold of sham-treated animals. When the effects of these opioids were examined with the tail-flick test using naive animals, oxycodone, morphine, and fentanyl exhibited antinociceptive effects on thermal nociception. These results show that the three opioids exhibit different efficacy outcomes in multiple pain models and that the efficacy profile of oxycodone does not overlap those of morphine and fentanyl.

Oxycodone-induced analgesic effects in a bone cancer pain model in mice.

The femur bone cancer pain model was developed by implanting mouse osteolytic tumor cells (NCTC 2472) into the intramedulla of the femur in C3H/HeN mice. In vivo imaging analysis revealed that the implanted tumor cells grew progressively over 14 days. Associated with the tumor growth, guarding behavior, which was an indication of ongoing pain, time-dependently increased. Limb use abnormality and allodynia, which were indications of ambulatory and neuropathic pain, respectively, also appeared. The analgesic effects of oxycodone and other opioids, such as morphine and fentanyl, were evaluated at 14 days when all pain-related behaviors clearly appeared. Oxycodone (2-20 mg/kg, s.c.), morphine (10-50 mg/kg, s.c.) and fentanyl (0.05-0.2 mg/kg, s.c.) significantly reduced guarding behavior. Oxycodone (5-20 mg/kg, s.c.) and fentanyl (0.1 and 0.2 mg/kg, s.c.) significantly reversed limb use abnormality, but morphine (5-50 mg/kg, s.c.) did not. Moreover, oxycodone (5-20 mg/kg, s.c.) dose-dependently reversed allodynia without affecting the sham-treated mice. Morphine (50 mg/kg, s.c.) and fentanyl (0.075-0.2 mg/kg, s.c.) also reversed allodynia, but morphine (50 mg/kg, s.c.) tended to affect and fentanyl (0.1 and 0.2 mg/kg, s.c.) affected the withdrawal threshold in sham-treated mice. These results suggested that oxycodone relieved not only ongoing pain, but also ambulatory and neuropathic pain, and that the analgesic profile of oxycodone could be different from that of either morphine or fentanyl.

Caffeic acid inhibits compound 48/80-induced allergic symptoms in mice.

The effect of caffeic acid on scratching behavior and vascular permeability changes induced by compound 48/80 in ICR mice were investigated. An oral dose of 500 mg/kg of caffeic acid significantly inhibited scratching behavior and vascular permeability induced by compound 48/80. The inhibitory effects of daily administration of lower doses of caffeic acid, 100 and 200 mg/kg, were also investigated; and it was found that 200 mg/kg significantly inhibited compound 48/80-induced scratching behavior after the second week of consecutive administration. The effect of 200 mg/kg of caffeic acid on scratching behavior was observed up to the third week of the treatment. The decrease in histamine content induced by compound 48/80 was significantly antagonized by 200 mg/kg. The findings suggest that caffeic acid may be effective for treating itch and edema in allergic dermatitis.

Effects of mometasone furoate on a rat allergic rhinitis model.

The present study was undertaken to clarify the effects of mometasone on nasal symptoms induced by repeated intranasal application of antigen in sensitized rats in comparison with that of chlorpheniramine. Rats received mometasone intranasally or chlorpheniramine orally 1 h before a topical antigen challenge for 7 days. Mometasone caused a decrease in the instances of nasal rubbing and an inhibition of this response was observed during the treatment period. Almost identical findings were observed with chlorpheniramine. This response was inhibited, even after the interruption of mometasone treatment, while such an effect was not observed with chlorpheniramine. On day 36, the changes in sensitivity to histamine were investigated. Unlike chlorpheniramine, hypersensitivity to histamine was significantly reduced in the mometasone-treated group. The passive cutaneous anaphylaxis titers were elevated and reached a maximum 8 days after the start of the topical antigen challenge. The passive cutaneous anaphylaxis titer in the mometasone-treated group was significantly lower than that in the control group. The results indicated that mometasone is effective in allergic rhinitis, not only during the period of application, but also after the interruption of application.

Increasing effect by simultaneous use of levocabastine and pemirolast on experimental allergic conjunctivitis in rats.

The effect of the simultaneous use of 0.025% levocabastine hydrochloride eye drops (levocabastine) and 0.1% pemirolast potassium ophthalmic solution (pemirolast) on experimental allergic conjunctivitis in rats was investigated. Levocabastine and pemirolast significantly inhibited allergic conjunctivitis compared with the control group when separately administered. In addition, the simultaneous use of both drugs inhibited allergic conjunctivitis more potently than the original activity of levocabastine or pemirolast. Furthermore, the simultaneous use of levocabastine and pemirolast also significantly inhibited increased vascular permeability induced by antigen compared with levocabastine or pemirolast alone, respectively. Levocabastine and pemirolast inhibited histamine release from the rat conjunctiva in correlation with a decrease in histamine content in tears. When levocabastine and pemirolast were simultaneously applied to the eyes, histamine release from the conjunctiva was greater than for the original activities of both drugs. Similar to histamine release from the conjunctiva, the histamine content in tears induced by the simultaneous use of both drugs was significantly decreased compared with levocabastine and pemirolast alone, respectively. A potentiating effect induced by the simultaneous use of levocabastine and pemirolast may be attributable to the antihistaminic activity of levocabastine and histamine release inhibition by levocabastine and pemirolast.

Participation of chemical mediators in the development of experimental allergic conjunctivitis in rats.

In the present study, we investigated the participation of chemical mediators in the development of experimental allergic conjunctivitis in rats. Cetirizine (a histamine H1 receptor antagonist), ramatroban (a thromboxane A2 (TXA2) receptor antagonist) and zafirlukast (a cysteinyl leukotrienes (cys-LTs) receptor antagonist) were orally administered from day 14 to day 42 during repeated topical antigen challenge. An increase in reactivity to antigen- and histamine-induced eye scratching behavior was observed by topical sensitization in sensitized rats. Although increased reactivity to antigen was not influenced by cetirizine, ramatoroban and zafirlukast, increased reactivity to histamine was significantly inhibited by ramatroban. The development of conjunctival edema was also observed for topical sensitization. Cetirizine caused no inhibition of the development of conjunctival edema, but ramatroban and zafirlukast inhibited the development of conjunctival edema. In addition, the number of eosinophils in the conjunctiva was increased by topical sensitization. Cetirizine had no significant effect on the increase in the number of eosinophils. However, ramatroban and zafirlukast were effective in inhibiting an increase in the number of eosinophils induced by topical sensitization. These results indicate that TXA2 is involved in increased histamine reactivity, and TXA2 and cys-LTs are associated with not only the conjunctival edema but also eosinophil infiltration during the development of experimental allergic conjunctivitis in rats.

A chronic model for evaluating the itching associated with allergic conjunctivitis in rats.

The present study was performed to develop a new model for evaluating itching associated with allergic conjunctivitis in rats. Repeated topical application of antigen caused an increase in eye scratching behavior in sensitized animals, and a significant difference was observed from days 21 to 42. Almost the same findings were observed in allergic symptoms, hyperemia and edema. Instillation of histamine also resulted in an increase in eye scratching behavior. The sensitivity to histamine in eye scratching behavior was increased by topical antigen application for 42 days after sensitization. In addition, the number of conjunctival eosinophils was significantly increased by repeated topical antigen application from days 21 to 42 in sensitized rats. Some anti-allergic drugs such as olopatadine (H1 antagonist), cetiridine (H1 antagonist) and ramatroban (thromboxane A2 (TXA2) antagonist) caused an inhibition of eye scratching behavior induced by topical sensitization in a dose-related manner. However, zafirlukast (cys-LT antagonist) caused no significant inhibition even at a dose of 30 mg/kg. The findings in present model of itching in allergic conjunctivitis were mainly through histamine H1-activity, and thromboxane A2 receptors were also involved in the response.