Naoyo Motoyama

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

Neuropathic pain is refractory against conventional analgesics, and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the regulation of pain signal transduction. Glycine transporter (GlyT)1, present in glial cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the neurons and thus could modify pain signal transmission in the spinal cord. In this study, we demonstrated that i.v. or intrathecal administration of GlyT1 inhibitors, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid (ORG25935) or sarcosine, and GlyT2 inhibitors, 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide (ORG25543) and (O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine) (ALX1393), or knockdown of spinal GlyTs by small interfering RNA of GlyTs mRNA produced a profound antiallodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The antiallodynia effect is mediated through spinal glycine receptor α3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. However, these manipulations to stimulate glycinergic neuronal activity were without effect during the 4 days after nerve injury, whereas manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of medication with their inhibitors should be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days after surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.

Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain

Injury to peripheral or spinal nerves following either trauma or disease has several consequences including the development of neuropathic pain. This syndrome is often refractory against conventional analgesics; and thus, novel medicaments are desired for its treatment. Recent studies have emphasized that dysfunction of inhibitory neuronal regulation of pain signal transduction may be relevant to the development of neuropathic pain. Glycinergic neurons are localized in specific brain regions and the spinal cord, where they play an important role in the prevention of pathological pain symptoms. Thus, an enhancement of glycinergic control in the spinal cord is a promising strategy for pain relief from neuropathic pain. Glycine transporter (GlyT) 1 and GlyT2, which are located in glial cells and neurons, respectively play important roles by clearing synaptically released glycine or supplying glycine to glycinergic neurons to regulate glycinergic neurotransmission. Thus, an inhibition of GlyTs could be used to modify pain signal transmission in the spinal cord. Recently developed specific inhibitors of GlyTs have made this possibility a reality. Both GlyT1 and GlyT2 inhibitors produced potential anti-nociceptive effect in various neuropathic pain models, chronic and acute inflammatory models in animals. Their anti-allodynia effects are mediated by the inhibition of GlyTs following activation of spinal glycine receptor alpha3. These results established GlyTs as target molecules for medicaments for neuropathic pain. Moreover, the phase-dependent anti-allodynia effects of GlyT inhibitors have provided important information on effective therapeutic strategies and also understanding the underlying molecular mechanisms of the development of neuropathic pain.

Pain-releasing action of Platelet-activating factor (PAF) antagonists in neuropathic pain animal models and the mechanisms of action

Platelet‐activating factor (PAF) has been implicated in the pathology of neuropathic pain. Previous studies reported that PAF receptor (PAF‐R) antagonists have varied anti‐allodynia effects by route of administration and nerve injury models in rats.

Relief of cancer pain by glycine transporter inhibitors.

BACKGROUND:Recent studies have revealed the antinociceptive effects of glycine transporter (GlyT) inhibitors in neuropathic pain models such as sciatic nerve–injured and diabetic animals. Bone cancer can cause the most severe pain according to complex mechanisms in which a neuropathic element is included. Bone cancer modifies the analgesic action of opioids and limits their effectiveness, and thus novel medicament for bone cancer pain is desired. METHODS:For the femur bone cancer model, NCTC 2472 tumor cells were injected into the medullary cavity of the distal femur of C3H/HeN mice. Effects of GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitors, ORG 25935, and knockdown of the expression of spinal GlyTs protein by GlyTs siRNA on pain-like behaviors, such as allodynia, withdrawal threshold, guarding behavior, and limb-use abnormality, were examined in the femur bone cancer model mice. Effects of morphine in combination with GlyT inhibitor were examined. RESULTS:GlyT2 inhibitors, ORG 25543 and ALX 1393, and GlyT1 inhibitor ORG 25935 by IV or oral administration or knockdown of the expression of spinal GlyTs protein improved pain-like behaviors at 11 days after tumor transplantation. The pain-relief activity was potent and long lasting. Morphine at a dose with no analgesic activity combined with ORG 25543 further promoted the ORG 25543–induced pain-relief activity. Injection of ORG 25543 on the second day after tumor implantation caused 3 phases of pain responses; pain-like behaviors were initially accelerated (at 2–4 days) and subsequently almost disappeared (5–7 days) and then reappeared. Intrathecal injection of strychnine 1 day after injection of ORG 25543 transiently antagonized the pain-relief activity of ORG 25543. In control mice, strychnine improved pain-like behaviors 4 days after tumor implantation and aggravated the behaviors between 4 and 5 days. The evidence suggests that the different mechanisms are phase-dependently involved. CONCLUSIONS:GlyT inhibitors with or without morphine may be a new strategy for the treatment of bone cancer pain and lead to further investigations of the mechanisms underlying the development of bone cancer pain.

Palliation of bone cancer pain by antagonists of platelet-activating factor receptors.

Bone cancer pain is the most severe among cancer pain and is often resistant to current analgesics. Thus, the development of novel analgesics effective at treating bone cancer pain are desired. Platelet-activating factor (PAF) receptor antagonists were recently demonstrated to have effective pain relieving effects on neuropathic pain in several animal models. The present study examined the pain relieving effect of PAF receptor antagonists on bone cancer pain using the femur bone cancer (FBC) model in mice. Animals were injected with osteolytic NCTC2472 cells into the tibia, and subsequently the effects of PAF receptor antagonists on pain behaviors were evaluated. Chemical structurally different type of antagonists, TCV-309, BN 50739 and WEB 2086 ameliorated the allodynia and improved pain behaviors such as guarding behavior and limb-use abnormalities in FBC model mice. The pain relieving effects of these antagonists were achieved with low doses and were long lasting. Blockade of spinal PAF receptors by intrathecal injection of TCV-309 and WEB 2086 or knockdown of the expression of spinal PAF receptor protein by intrathecal transfer of PAF receptor siRNA also produced a pain relieving effect. The amount of an inducible PAF synthesis enzyme, lysophosphatidylcholine acyltransferase 2 (LPCAT2) protein significantly increased in the spinal cord after transplantation of NCTC 2472 tumor cells into mouse tibia. The combination of morphine with PAF receptor antagonists develops marked enhancement of the analgesic effect against bone cancer pain without affecting morphine-induced constipation. Repeated administration of TCV-309 suppressed the appearance of pain behaviors and prolonged survival of FBC mice. The present results suggest that PAF receptor antagonists in combination with, or without, opioids may represent a new strategy for the treatment of persistent bone cancer pain and improve the quality of life of patients.

Antinociceptive effects of glycine transporter inhibitors in neuropathic pain models in mice.

神経因性疼痛は難治性で従来の鎮痛薬が奏効せず，新しい薬が期待されている．グリシン作動性ニューロンは脊髄を含む脳の特定部位に局在し，痛みシグナルの伝達を調節する重要な役割を果たしている．本研究では，坐骨神経部分結紮（PSL）神経因性疼痛モデルマウスを用いてグリシントランスポーター（GlyT）阻害薬の抗侵害作用について検討し，特異的GlyT1阻害薬ORG25935，sarcosineおよびGlyT2阻害薬ORG25543，ALX1393の脊髄腔内投与および静脈内投与によりアロディニア症状を寛解することを見い出した．この作用は強力で長期間持続し，gabapentinの作用よりも著しく長いものであった．GlyTs阻害薬の抗アロディニア作用はGlyTsの阻害によるグリシンの貯留と，それに続く脊髄グリシン受容体α3（GlyRα3）の活性化を介したグリシン作動性抑制系の賦活によることを示した．グリシン神経の活性化は神経損傷の初期では侵害的に作用し，4日以降に抗侵害作用を認めた．この逆転現象はミクログリアの活性化，脳由来神経栄養因子（BDNF），細胞内Cl-の汲み出しに機能するKCC2の発現抑制といった一連のカスケードが関与する可能性を示唆した．以上，GlyTs阻害薬に強力な抗アロディニア作用を認め，GlyTは神経因性疼痛の治療薬開発のターゲットとなることを示した．更に，GlyTs阻害薬の作用は，神経損傷後3～4日を境に逆転した．このことよりGlyT阻害薬は投与のタイミングが重要であることを明らかにした．また，この逆転現象は神経因性疼痛発症の基盤となる分子メカニズムを解明する面からも興味がもたれる．