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Opioid-induced constipation (OIC), a typical side effect of opioids, is due to activation of the μ-opioid receptors in the enteric nervous system. Peripherally acting μ-opioid receptor antagonists (PAMORAs) can reverse OIC by inhibiting the peripheral action of opioids without affecting centrally mediated analgesia. Naldemedine is a PAMORA with potent antagonist activity against μ-, δ-, and κ-opioid receptors. In this study, the pharmacological profiles of naldemedine, compared with those of naloxone and naloxegol, were evaluated. In vitro, Schild plot analysis indicated that naldemedine was a noncompetitive antagonist of μ-opioid receptors, whereas other compounds were competitive antagonists. Also, naldemedine showed slower association and dissociation kinetics than the other compounds. In vivo, naldemedine dose-dependently ameliorated morphine-induced inhibition of small intestinal transit (SIT). The dose-response curve was not shifted at 1 and 3 mg/kg morphine. On the contrary, that of naloxegol was significantly shifted to the right from 1 to 3 mg/kg morphine. In morphine-dependent rats, naldemedine caused peripheral withdrawal symptoms (diarrhea) at doses higher than 1 mg/kg, whereas the dose that produced half the maximal preventive effect (ED50) against constipation was 0.03 mg/kg. Naldemedine showed slower onset and a lesser severity of diarrhea than the other compounds at close to the ED50 value in the SIT model. Our results reveal that naldemedine has different pharmacological profiles (type of antagonism and binding kinetics) to the other compounds. This might explain the differential inhibition of morphine-induced SIT and withdrawal symptoms among the three antagonist compounds. SIGNIFICANCE STATEMENT: Naldemedine is a novel peripherally acting μ-opioid receptor antagonist with potent antagonist activity against μ-, δ-, and κ-opioid receptors. Naldemedine showed a noncompetitive antagonism and slower association and dissociation kinetics against μ-opioid receptors than naloxone and naloxegol. Naldemedine showed insurmountable antagonism of morphine-induced inhibition and lower and slower peripheral withdrawal symptoms (diarrhea) than the other compounds. Therefore, naldemedine has a different pharmacological profile (the type of antagonism and binding kinetics) to the other compounds.

Structure-activity relationship studies of several morphinan derivatives were conducted to obtain dual antagonists for μ- and δ-opioid receptors. We discovered peripherally restricted dual antagonists for μ/δ-opioid receptors as a new chemotype with a morphinan scaffold, which are orally available and do not easily pass the blood–brain barrier. As we expected, some of these compounds inhibit opioid-induced constipation and emesis/vomiting with limited potential to interfere the analgesic effects of morphine. Among them, naldemedine was selected as a potential drug candidate.

Naloxone is a μ-opioid receptor antagonist that has been used to prevent overdose-related respiratory depression and deaths by the illicit use of opioids. Naloxone can also deter the abuse potential of opioids, but little has been reported regarding its antagonistic activity profile against opioid-induced psychological dependence. This study aimed to confirm the antagonistic activity profile of naloxone against several μ-opioid receptor agonists and investigate whether naloxone could affect the psychological dependence induced by widely used μ-opioid receptor agonist, oxycodone. In the Guanosine-5’-o-(3-thio) triphosphate (GTPγS) binding assay, naloxone (30−30,000 nM) inhibited the GTPγS binding induced by oxycodone, hydrocodone, morphine, and fentanyl. It elicited parallel rightward shifts in the concentration-response curves, indicating that naloxone possessed a competitive antagonistic activity profile against these μ-opioid receptor agonists. In the conditioned place preference test, oxycodone (0.01−1 mg/kg, i.v.) produced dose-dependent increases in place preference. The increased place preference induced by oxycodone (1 mg/kg) was significantly attenuated by co-administration of naloxone at a dose of 0.5 mg/kg but not 0.01 mg/kg. Naloxone (0.5 mg/kg, i.v.) also blocked oxycodone (1 mg/kg)-induced dopamine release in nucleus accumbens; however, at a lower dose (0.01 mg/kg), it did not affect the intrinsic dopamine release by oxycodone. These results indicate that the psychological dependence of oxycodone could be antagonized by naloxone, depending on the dose. This characterization might lead to a better understanding of the competitive antagonistic activity profile of naloxone for μ-opioid receptor in the brain.

Opioids are widely used for pain management in moderate-to-severe pain. However, opioids are associated with adverse events, such as constipation and emesis/vomiting. To reduce these undesired effects, a structure–activity relationship study of morphinan derivatives was conducted, and a promising lead compound with inhibitory effects on opioid receptors was obtained. Further improvement in the potency and pharmacokinetic profiles of the lead compound led to the discovery of naldemedine, which showed anti-constipation and anti-emetic effects against these adverse events that were induced by morphine without influencing morphine’s analgesic effect. Naldemedine was launched in Japan and the USA in 2017 and in the EU in 2019, for treating opioid-induced constipation.

Opioid-induced constipation (OIC), a typical side effect of opioids, is due to activation of the

A novel series of (6-aminopyridin-3-yl)(4-(pyridin-2-yl)piperazin-1-yl) methanone derivatives were identified as selective transient receptor potential vanilloid 4 (TRPV4) channel antagonist and showed analgesic effect in Freund's Complete Adjuvant (FCA) induced mechanical hyperalgesia model in guinea pig and rat. Modification of right part based on the compound 16d which was disclosed in our previous communication led to the identification of compound 26i as a flagship compound. In this paper, we described the details about design, synthesis and structure-activity relationship (SAR) analysis at right and left part of these derivatives (Fig. 1).

Background Naldemedine (S‐297995) is a peripherally acting μ‐opioid receptor antagonist developed as a once‐daily oral drug for opioid‐induced constipation (OIC) in adults with chronic noncancer or cancer pain. This study characterized the pharmacological effects of naldemedine in vitro and in vivo. Methods The binding affinity and antagonist activity of naldemedine against recombinant human μ‐, δ‐, and κ‐opioid receptors were assayed in vitro. Pharmacologic effects of naldemedine were investigated using animal models of morphine‐induced inhibition of small and large intestinal transit, castor oil‐induced diarrhea, antinociception, and morphine withdrawal. Key Results Naldemedine showed potent binding affinity and antagonist activities for recombinant human μ‐, δ‐, and κ‐opioid receptors. Naldemedine significantly reduced opioid‐induced inhibition of small intestinal transit (0.03‐10 mg kg−1; P, Naldemedine displayed potent binding affinity to, and antagonistic activity against, μ‐, δ‐, and κ‐opioid receptors. Naldemedine tempered OIC in vivo without compromising opioid analgesia.

A novel series of 2',4'-dimethyl-[4,5'-bithiazol]-2-yl amino derivatives were found by high throughput screening of the TRPV4 receptor, at which these compounds showed competitive antagonist potential against 4α-phorbol 12,13-didecanoate (4αPDD) as the selective TRPV4 agonist and showed excellent selectivity for TRPV1, N-type and L-type calcium ion channels, but poor ADME profile. In our SAR strategy, we found that the lead molecule 1 also having the unique 3-oxa-9-azabicyclo [3.3.1] nonan-7-one on the right part showed potent TRPV4 antagonist activity, good solubility at pH 6.8, good microsomal stability for human and better ADME profile including oral bioavailability. Moreover, compound 1 had an analgesic effect in Freund's Complete Adjuvant (FCA) induced mechanical hyperalgesia model in guinea pig. In this letter, we report a lead optimization process to identify the lead compound 1 (Fig. 1).

Aims Naldemedine is a peripherally acting μ-opioid receptor antagonists (PAMORAs) indicated for the treatment of opioid-induced constipation (OIC). We investigated the preventive effect of naldemedine on morphine-induced nausea and vomiting in ferrets and conducted a pharmacokinetic/pharmacodynamic (PK/PD) analysis. Main methods The antiemetic effect of naldemedine was evaluated as the frequency and time of retching (rhythmic abdominal contractile motion) and vomiting (throwing up vomit or similar reactions) caused by morphine in ferrets. After a single oral administration of naldemedine to ferrets, the plasma concentrations of naldemedine and morphine were measured by liquid chromatography–tandem mass spectrometry. Key findings Naldemedine showed a potent and dose-dependent anti-emetic effects against morphine-induced emetic responses, for up to 6 h. The dose of naldemedine that produced half the maximal effect (ED50) value for anti-emetic effect of naldemedine in the morphine-treated ferrets was 0.033 mg/kg. The PK/PD analysis revealed that the antiemetic effect was related to the plasma naldemedine concentration, with a half maximal effective concentration that produces half the maximal effect (EC50) of 3.51 ng/mL. The plasma concentration producing an antiemetic effect was almost 200-fold lower than that inducing an anti-analgesic effect in rats. Significance Naldemedine showed potent inhibition of morphine-induced vomiting for up to 6 h after dosing. These data suggest that naldemedine possesses antiemetic properties and could be effective against opioid-induced nausea and vomiting (OINV).

Transient receptor potential vanilloid 4 (TRPV4) receptor modulates pain, and this has been noted in several animal models. However, the involvement of TRPV4 in osteoarthritic (OA) pain remains poorly understood. This study assessed the functional changes in TRPV4 and the expression of its endogenous ligand 5,6-epoxyeicosatrienoic acid (5,6-EET) in a rat monoiodoacetate (MIA)-induced OA pain model (MIA rats). Monoiodoacetate-treated rats showed reduced grip strength as compared to sham-treated rats, and this loss in function could be recovered by the intraarticular administration of a TRPV4 antagonist (HC067047 or GSK2193874). By contrast, the intraarticular administration of the TRPV4 agonist, GSK1016790A, increased the pain-related behaviors in MIA rats but not in sham rats. TRPV4 expression was not increased in knee joints of MIA rats; however, the levels of phosphorylated TRPV4 at Ser824 were increased in dorsal root ganglion neurons. In addition, 5,6-EET was increased in lavage fluids from the knee joints of MIA rats and in meniscectomy-induced OA pain model rats. 5,6-EET and its metabolite were also detected in synovial fluids from patients with OA. In conclusion, TRPV4 was sensitized in the knee joints of MIA rats through phosphorylation in dorsal root ganglion neurons, along with an increase in the levels of its endogenous ligand 5,6-EET. The analgesic effects of the TRPV4 antagonist in the OA pain model rats suggest that TRPV4 may be a potent target for OA pain relief.

It has begun to be understood that μ-opioid receptor (MOR) produces ligand-biased agonism, which contributes to differential physiological functions of MOR agonists. We previously demonstrated that in oxaliplatin-induced neuropathy in rats, morphine and oxycodone exhibited antinociceptive effects while antinociception of fentanyl was partial, and such different efficacies might result from the different level of Gi/o protein activation. Based on our background, to reveal further mechanism, we focused on the role of Gi/o protein-related downstream signaling, the G-protein inwardly rectifying K(+)1 (GIRK1) channel. The GIRK1 channel blocker tertiapin-Q (30pmol) was intracerebroventricularly (i.c.v.) or intrathecally (i.t.) administered to rats with oxaliplatin-induced neuropathy. The antinociception of systemic morphine (3mg/kg, subcutaneously (s.c.)) was suppressed only by pretreatment of i.t. tertiapin-Q, while supraspinal tertiapin-Q suppressed only the antinociception of systemic oxycodone (0.56mg/kg, s.c.). Partial antinocicpetion of fentanyl (0.017mg/kg, s.c.) was neither affected by i.c.v nor i.t. tertiapin-Q. These results demonstrated that GIRK1 channels differentially contribute to antinociceptive effects of MOR agonists, and that action site of GIRK1 channels is also different between morphine and oxycodone in oxaliplatin model. This study suggests the possibility that GIRK1 channels have a crucial role for antinociception of MOR agonists in oxaliplatin-induced neuropathy.

A series of novel tetrahydropyridinecarboxamide TRPV1 antagonists were prepared and evaluated in an effort to optimize properties of previously described lead compounds from piperazinecarboxamide series. The compounds were evaluated for their ability to block capsaicin and acid-induced calcium influx in CHO cells expressing human TRPV1. The most potent of these TRPV1 antagonists were further characterized in pharmacokinetic, efficacy, and body temperature studies. On the basis of its pharmacokinetic, in vivo efficacy, safety, and toxicological properties, compound 37 was selected for further evaluation in human clinical trials.

Oxaliplatin is a chemotherapeutic agent that induces chronic refractory neuropathy. To determine whether opioids effectively relieve this chronic neuropathy, we investigated the efficacies of morphine, oxycodone, and fentanyl, and the mechanisms underlying opioid antinociception, in oxaliplatin-induced neuropathy in rats. Rats exhibited significant mechanical allodynia following 2 weeks of chronic oxaliplatin administration. Within the range of doses that did not induce sedation and/or muscle rigidity, morphine (3 mg/kg, subcutaneously, s.c.) and oxycodone (0.3 – 0.56 mg/kg, s.c.) completely reversed oxaliplatin-induced mechanical allodynia, whereas fentanyl (0.017 – 0.03 mg/kg, s.c.) showed partial antinociception. The antinociception of the optimal doses of morphine and oxycodone were completely inhibited by pertussis toxin (PTX; 0.5 μg/rat, i.c.v.), a Gi/o protein inhibitor, while the partial effect of fentanyl was not affected in the oxaliplatin model. In the [35S]-GTPγS binding assay, activation of μ-opioid receptor by fentanyl, but not by morphine or oxycodone, in the mediodorsal thalamus was significantly reduced in oxaliplatin-treated rats. These results indicate that the lower antinociceptive potency of fentanyl in the oxaliplatin model might in part result from the loss of PTX-sensitive Gi/o protein activation, and the degree of Gi/o protein activation might be related to the potency of antinociception by opioids in this model. Keywords:: opioid, antinociception, oxaliplatin, neuropathy, Gi/o protein

The rewarding effects of μ-receptor agonists can be suppressed under several pain conditions. We recently showed that clinically used μ-receptor agonists possess efficacies for relieving the neuropathic pain induced by chemotherapeutic drug in rats; however, it is possible that the use of μ-receptor agonists may trigger the rewarding effects even under chemotherapeutic drug–induced neuropathic pain. Nevertheless, no information is available regarding whether μ-receptor agonists produce psychological dependence under chemotherapeutic drug–induced neuropathic pain. Therefore, we examined the effects of neuropathy induced by chemotherapeutic drugs on the rewarding effects of morphine, oxycodone, and fentanyl in rats. Repeated treatment with oxaliplatin or paclitaxel produced neuropathy as measured by the von Frey test. Rewarding effects produced by antinociceptive doses of μ-receptor agonists were not suppressed under oxaliplatin- or paclitaxel-induced neuropathy. Furthermore, the morphine-induced increase in the release of dopamine from the nucleus accumbens, which is a critical step in the rewarding effects of μ-receptor agonists, was not altered in paclitaxel-treated rats. These results suggest that the rewarding effects of μ-receptor agonists can still be established under oxaliplatin- or paclitaxel-induced neuropathic pain. Therefore, patients should be carefully monitored for psychological dependence on μ-receptor agonists when they are used to control chemotherapeutic drug-induced neuropathic pain. Keywords:: rewarding effect, oxaliplatin, paclitaxel, neuropathic pain, μ-opioid receptor agonist

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 antinociceptive 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 α1-adrenoceptor antagonists. The partial anti-nociceptive effect of tapentadol (30 mg/kg) was completely suppressed by a MOR antagonist, but not by α1- or α2-adrenoceptor antagonists, suggesting that neither α1-adrenoceptor- nor α2-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. Keywords:: femur bone cancer pain, norepinephrine transporter, oxycodone, tapentadol, μ-opioid receptor (MOR)

A series of 2',4'-dimethyl-[4,5'-bithiazol]-2-yl amino derivatives have been identified as selective TRPV4 antagonists that display inhibition potencies against 4α-phorbol 12,13-didecanoate (4αPDD), well known as a TRPV4 selective agonist and/or a hypotonicity. In particular, 9-(6-((2',4'-dimethyl-[4,5'-bithiazol]-2-yl)amino)nicotinoyl)-3-oxa-9-azabicyclo[3.3.1]nonan-7-one showed an analgesic effect in Freund's Complete Adjuvant (FCA) induced mechanical hyperalgesia model in guinea pig (reported in Part 1). However, there are some concerns such as species differences and the need for higher plasma exposure to achieve target efficacy for evaluation by an in vivo pain model. In this Letter, we report the resolution of some of the problems by further optimizing the chemical scaffold.

Although estrous cycle has been reported to influence antiociceptive effect of morphine in several pain conditions, its effect on cancer pain is not well established. We investigated the effect of estrogen on morphine antinociception using a bone cancer pain model and compared its potency with that of oxycodone. Female mice were ovariectomized (OVX) for preparation of a femur bone cancer pain (FBC) model. β-estradiol was subcutaneously (s.c.) administered and antinociceptive effects of opioids was assessed using the von Frey monofilament test. Although morphine (5-20mg/kg, s.c.) did have significant antinociceptive effects in the FBC-OVX group, its effects in the FBC-OVX+β-estradiol (OVX+E) group was limited. Oxycodone (1-5mg/kg, s.c.) exhibited significant effects in both groups. Expression changes in opioid-related genes (μ-, κ-, δ-opioid receptors, prodynorphin, proenkephalin, proopiomelanocortin) in the spinal and supraspinal sites were examined among the sham-OVX, sham-OVX+E, FBC-OVX, and FBC-OVX+E groups by in situ hybridization. These studies detected a significant increase in prodynorphin in the spinal dorsal horn of the FBC-OVX+E group. Spinal injection of a dynorphin-A antibody to FBC-OVX+E mice restored antinociception of morphine. In conclusion, we detected a differential effect of estrogen on morphine- and oxycodone-induced antinociception in a female FBC model. The effect of morphine was limited with estrogen exposure, which may be due to estrogen- and pain-mediated spinal expression of dynorphin-A.

In addition to pain sensation, both thermal regulation and thermosensation are thought to be influenced by TRPV1 channel functioning. Involvement of TRPV1 in these latter physiological functions became clear during pharmaceutical development of selective TRPV1 antagonists for the treatment of chronic painful conditions. The majority of TRPV1 antagonists that have been progressed into clinical trials were reported to increase body temperature (hyperthermia) in healthy humans. Currently, hyperthermia is a major concern in pharmaceutical development of TRPV1 antagonists. We therefore sought of an in-vitro method to identify compounds with a low hyperthermic potential early in the drug discovery process. Using whole-cell patch clamp electrophysiology on human TRPV1 expressing CHO cells, we performed kinetic measurements of dissociation from the receptor of known hyperthermic (AMG517 and ABT102) and non-hyperthermic (SB705498) TRPV1 antagonists. We found that AMG517 and ABT102, two hyperthermic compounds, exhibited slow off- (dissociation) rates when channels were activated by capsaicin. Conversely, the non-hyperthermic compound SB705498, showed a significantly faster rate of dissociation from the receptor. Finally, we assessed an in-house proprietary antagonist V116517 which demonstrated significant separation between analgesic efficacy and hyperthermia. Once again, this compound displayed a faster off-rate as compared to the hyperthermic compounds.With these initial findings we hypothesize that the dissociation rate of an antagonist from the TRPV1 channel is an important parameter in determining a compound's effect on thermal regulation and thermosensation. Antagonists with a fast off-rate may allow endogenous ligands to interact with the TRPV1 channel and thereby fulfil its physiological role in body temperature regulation while still effectively modifying pain signaling. In conclusion, our findings suggest that in vitro kinetic measurements early in the drug discovery process may be a useful means of identifying non-hyperthermic TRPV1 antagonists.

A series of novel tetrahydropyridinecarboxamide TRPV1 antagonists were prepared and evaluated in an effort to optimize properties of previously described lead compounds from piperazinecarboxamide series. The compounds were evaluated for their ability to block capsaicin and acid-induced calcium influx in CHO cells expressing human TRPV1. The most potent of these TRPV1 antagonists were further characterized in pharmacokinetic, efficacy, and body temperature studies. On the basis of its pharmacokinetic, in vivo efficacy, safety, and toxicological properties, compound 37 was selected for further evaluation in human clinical trials.

In this study, we investigated the effect of alpha-eudesmol, which potently inhibits the presynaptic omega-agatoxin IVA-sensitive (P/Q-type) Ca(2+) channel, on neurogenic inflammation following electrical stimulation of rat trigeminal ganglion. Treatment with alpha-eudesmol (0.1-1 mg/kg. i.v.) dose-dependently attenuated neurogenic vasodilation in facial skin monitored by a laser Doppler flowmetry. In addition, alpha-eudesmol (1 mg/kg. i.v.) significantly decreased dural plasma extravasation in analysis using Evans blue as a plasma marker. On the other hand, alpha-eudesmol (1 mg/kg, i.v.) did not affect mean arterial blood pressure in rats. The calcitonin gene-related peptide (CGRP) and substance P (SP) released from activated sensory nerves have recently been suggested to be associated with the neurogenic inflammation. In this study, we also showed that alpha-eudesmol (0.45-45 microM) concentration-dependently inhibits the depolarization-evoked CGRP and SP release from sensory nerve terminals in spinal cord slices. These results indicate that the anti-neurogenic inflammation action of alpha-eudesmol, which does not affect the cardiovascular system, may be due to its presynaptic inhibition of the neuropeptide release from perivascular trigeminal terminals. We also suggest that the omega-agatoxin IVA-sensitive Ca(2+) channel blocker, alpha-eudesmol, may become useful for the treatment of the neurogenic inflammation in the trigemino-vascular system such as migraine.

omega-Agatoxin IVA-sensitive Ca(2+) channels have been thought to be involved in physiological excitatory amino acid glutamate release and these channels may also contribute to the development of ischemic brain injury. Recently, we demonstrated that alpha-eudesmol from Juniperus virginiana Linn. (Cupressaceae) inhibits potently the presynaptic omega-agatoxin IVA-sensitive Ca(2+) channels. In the present study, we investigated the effects of alpha-eudesmol on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery occlusion in rats. We first found that alpha-eudesmol concentration-dependently inhibited glutamate release from rat brain synaptosomes and that its inhibitory effect was Ca(2+)-dependent. In the middle cerebral artery occlusion study, intracerebroventricular (i.c.v.) treatment with alpha-eudesmol significantly attenuated the post-ischemic increase in brain water content. alpha-Eudesmol also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. Using a microdialysis technique, we further demonstrated that alpha-eudesmol inhibits the elevation of the extracellular concentration of glutamate during ischemia. From these results, we suggest that alpha-eudesmol displays an ability to inhibit exocytotic glutamate release and to attenuate post-ischemic brain injury.

Recently, the omega-agatoxin IVA (omega-Aga-IVA)-sensitive Ca2+ channel has been demonstrated to play an important role in the physiological neurotransmitter release in mammalian nerve terminals. In this study, we demonstrate that alpha-eudesmol from Juniperus virginiana Linn. (Cupressaceae) inhibits omega-Aga-IVA-sensitive Ca2+ channels in rat brain synaptosomes and cerebellar Purkinje cells. Thirty millimolar KCl-induced 45Ca2+ uptake into the synaptosomes was inhibited by omega-Aga-IVA but insensitive to omega-conotoxin GVIA (omega-CTX-GVIA, N-type Ca2+ channel blocker) and nicardipine (L-type Ca2+ channel blocker). We found that alpha-eudesmol concentration-dependently inhibited the above synaptosomal 45Ca2+ uptake with an IC50 value of 2.6 microM. Co-treatment with alpha-eudesmol and omega-Aga-IVA did not cause any additive inhibitory effect against the synaptosomal 45Ca2+ uptake. Using the whole-cell patch clamp electrophysiological technique, we further demonstrated that alpha-eudesmol concentration-dependently inhibited omega-Aga-IVA-sensitive Ca2+ channel currents recorded from Purkinje cells with an IC50 value of 3.6 microM. The current-voltage relationship of the omega-Aga-IVA-sensitive Ca2+ channel currents was not changed by alpha-eudesmol. On the other hand, alpha-eudesmol also displayed an inhibitory effect on N-type Ca2+ channel currents recorded from differentiated NG108-15 cells with an IC50 value of 6.6 microM. However, alpha-eudesmol had little inhibitory effect on L-type Ca2+ channel currents. Thus, the present data indicated that alpha-eudesmol is a potent nonpeptidergic compound which blocks the presynaptic omega-Aga-IVA-sensitive Ca2+ channel with relative selectivity.

The previously reported morphinan derivative SN-28 showed high selectivity and agonist activity for the δ opioid receptor. In the course of examining the structure–activity relationship of SN-28 derivatives, the derivatives with the 4-hydroxy group (SN-24, 26, 27) showed higher selectivities for the δ receptor over the μ receptor than the corresponding SN-28 derivatives with the 3-hydroxy group (SN-11, 23, 28). Derivatives with the 4-hydroxy group showed potent agonist activities for the δ receptor in the [35S]GTPγS binding assay. Although the 17-cyclopropylmethyl derivative (SN-11) with a 3-hydroxy group showed the lowest selectivity for the δ receptor among the morphinan derivatives, the agonist activity toward the δ receptor was the most potent for candidates with the 3-hydroxy group.

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. Keywords:: oxycodone, morphine, fentanyl, neuropathic pain-like state, bone cancer pain

We re-examined the accessory site of the 4,5-epoxymorphinan skeleton by camdas conformational analysis in an effort to deign novel delta opioid receptor antagonists. We synthesized three novel compounds (SN-11, 23 and 28) with a 10-methylene bridge and without a 4,5-epoxy ring. Among them, compounds SN-23 (17-isobutyl derivative) and SN-28 (17-methyl derivative) showed very strong agonist activity (over 10 times more than TAN-67). SN-28 also showed high delta selectivity. The delta agonist activity of SN-23 was weaker than that of SN-28, but in terms of the delta selectivity, SN-23 was higher than that of SN-28. These unexpected results indicated that the 4,5-epoxy ring, but not the 10-methylene bridge, was an accessory site in delta opioid receptor agonists.

Structure–activity relationships and efforts to optimize the pharmacokinetic profile of a class of 2-arylimino-5,6-dihydro-4H-1,3-thiazines as cannabinoid receptor agonists are described. Among the compounds examined, compound 14 showed potent affinity and high selectivity for CB2, and compound 23 showed potent affinities against CB1 and CB2. These compounds displayed oral bioavailability.

We examined the behavioral pharmacological properties of six benzodiazepine (omega) receptor ligands including brotizoram, nitrazepam, quazepam, rilmazafone, zolpidem and zopiclone and the binding of these drugs with omega receptor subtypes. Behavioral tests were performed at the time of the maximal effects induced by each drug following its oral administration to mice. All of these drugs dose-dependently induced impairment of motor coordination as rotarod performance and potentiation of thiopental-induced anesthesia as hypnotic effect. The hypnotic effects of rilmazafone, whose major metabolites were bound to both omega1 and omega2 receptors with high affinity, and omega1 selective quazepam were about 20 times more effective than the induction of motor impairments when compared with ED50 values. However, there was no difference between the ED50 values of omega1 selective zolpidem alone in these two tests. An antianxiety efficacy of zolpidem was relatively weak unlike that of other drugs in the elevated plus-maze. It has been reported that omega2, but not omega1, receptors are associated with motor impairment and anxiolytic effect. The weak anxiolytic effect of zolpidem supports the previous hypothesis. However, the strong motor incoordination of zolpidem suggests that not only omega2 but also omega1 receptors are related to motor impairment unlike the previous hypothesis.

The synthesis and structure activity relationships of a series of N-pyrimidinyl benzenesulfonamides as ETB selective antagonists are described. N-Isoxazolyl benzenesulfonamide 1a, previously reported, (1) was selected as a lead compound, and isosteric replacement of the isoxazole ring of 1a with a pyrimidine ring led to the discovery of the highly potent ETB selective antagonist 6e with oral bioavailability. Modification of the terminal aldehyde group at the 6-position of the pyrimidine ring was investigated, and malonate 15b and acylhydrazone 16f were found to be equipotent to aldehyde 6e. Compound 6e showed ETB antagonistic activity on in vivo evaluation.

Recently, P/Q-type Ca2+ channels have been shown to be involved in neurotransmission in the central nervous system in mammals. We evaluated the effects of the P/Q-type Ca2+ channel blocker omega-agatoxin IVA (omega-Aga-IVA) on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery (MCA) occlusion in rats. Intracerebroventricular (i.c.v.) treatment with omega-Aga-IVA significantly attenuated the postischemic increase of brain water content. omega-Aga-IVA also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. omega-Aga-IVA (30 pmol, i.c.v.), which exhibited a neuroprotective effect, had no significant effect on the magnitude of intra- and postischemic brain temperature when compared with vehicle-treated rats. This indicates that the postischemic neuroprotective effect of omega-Aga-IVA is produced by a direct and not an indirect effect via hypothermia. These results suggest that P/Q-type Ca2+ channels may be involved in the development of focal ischemic brain injury and that blockers of these channels may be therapeutically useful against ischemic injury.

The effects of U50488, kappa-opioid agonist on P-type Ca2+ channels, were studied. U50488 inhibited depolarization-induced Ca2+ uptake into rat brain synaptosomes, which was sensitive to omega-Agatoxin IVA (omega-AgaIVA; P-type Ca2+ channel blocker) and inhibited P-type Ca2+ channel currents recorded from rat cerebellar Purkinje neurons by the whole-cell patch clamp method. Dynorphin A also inhibited P-type Ca2+ channel currents. The inhibition by U50488 was biphasic; high affinity component (21%, IC50 = 8.9 x 10(-8) M) and low affinity component (79%, IC50 = 1.1 x 10(-5) M). At low concentrations of U50488 (10(-6) M), P-type Ca2+ channel current inhibition was attenuated by norbinartorphimine (nor-BNI), kappa-opioid antagonist, and by dialysis of cells with a pipette solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP-beta S). At high concentrations of U50488 (10(-5) M), P-type Ca2+ channel current inhibition was frequency-dependent. Thus U50488-induced current inhibition is mediated by two mechanisms. Its high affinity component is produced by activation of kappa-opioid receptors, whereas the low affinity component is due to its direct action on the P-type Ca2+ channel.

Pancreatic-type group I phospholipase A2 (PLA2-I), EC 3.1.1.4, long thought to act as a digestive enzyme, has a specific binding site in several types of tissues and cells and these sites promote PLA2-I-stimulated DNA synthesis. In this study we report a PLA2-I action on the migration of rat embryonic thoracic aorta smooth muscle cells (A7r5). A7r5 cells had a single class of PLA2-I binding site with an equilibrium binding constant (Kd) value of 1.7 nM and a maximum binding capacity (Bmax) of 40,000 sites/cell. The migration activity of PLA2-I for A7r5 cells was examined using modified Boyden chambers. PLA2-I stimulated the migration dose-dependently, and the ED50 value was about 1 nM, which was almost the same as the Kd value for PLA2-I binding. Checkerboard analysis showed that the response of A7r5 cells to PLA2-I was chemokinetic, but not chemotactic. These findings reveal a new aspect of PLA2-I in the modulation of vascular function.

The effects of nucleotides on the Ca2+-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx was measured by following the change in scattered light intensity using the stopped flow technique. ATP enhanced the Ca2+-induced choline influx. The activation followed a single-site titration curve with a dissociation constant of 1.0 +/- 0.5 mM, independent of the Ca2+ concentration. ATP seems to increase the pore radius or number of channels without affecting the gating mechanism of the Ca2+-gated cation channel. ADP, AMP, and adenine enhanced the choline transport in a manner similar to ATP, but cAMP, ITP, UTP, CTP, and GTP did not. The apparent dissociation constants and the maximal activations were as follows: ATP 1.0 mM, 28-fold; ADP 0.9 mM, 18-fold; AMP 0.6 mM, 7-fold, and adenine 0.4 mM, 4-fold. Adenine and AMP behaved as a competitive inhibitor for the activation by ATP. These results are consistent with the Ca2+-induced Ca2+ release observed in skinned muscle fiber and isolated SR.

Porcine pancreatic group I phospholipase A2 (PLA2-I) induced contraction of guinea pig lung parenchyma in a concentration-dependent manner. Its EC50 value was similar to theKd value calculated from the specific binding of125I-labeled porcine PLA2-I in the membrane fraction of guinea pig lung. Type-specific action of PLA2's and homologous desensitization strongly implicated the involvement of PLA2-I-specific sites in the activation process. Throm☐ane A2 was found to be the main product from lung tissue by PLA2-I action and the contractile response by PLA2-I was specifically suppressed by throm☐ane A2 receptor antagonists and cyclooxygenase inhibitor, but not by leukotriene receptor antagonist and H1 blocker. These findings indicate that PLA2-I-induced contractile response may depend on the secondarily produced throm☐ane A2, thus providing a new aspect of PLA2-I from the pathophysiological standpoint.

Osmotic volume change of sarcoplasmic reticulum vesicles was studied by following the change in light-scattering intensity using a stopped flow apparatus. From the analysis of the initial rate of scattering change, reflection coefficients for various ions and neutral molecules were determined. The following are typical results: K+, 0.72; Tris+, 0.98; choline 1; NO3-, 0.32; Cl-, 0.46; methanesulfonate, 0.62; gluconate, 0.96; glycerol, 0.86; and glucose, 1. When the K+ permeability was increased in the presence of 10(-6) g valinomycin/ml, the reflection coefficient for K+ changed from 0.72 to 0.31. It was found that there was a close relationship between the reflection coefficients and the permeabilities of the solutes. Hydraulic conductivity was also determined from the initial rate of light scattering change and was not different for the different solutes. The water permeability was estimated to be 2.1 x 10(-3) cm/sec at 23 degrees C.