瞬时感受器电位离子通道香草素受体4下游信号分子的确定及其对大鼠背根神经节慢性压迫后痛觉过敏的作用

目的:探讨大鼠背根神经节慢性压迫CCD后瞬时感受器电位离子通道香草素受体4(TRPV4)下游信号分子及其在痛觉过敏中的机制。方法:鞘内分别注射TRPV4拮抗剂钌红(RR)、TRPV4反义寡脱氧核苷酸(ASODN)和一氧化氮合成酶(NOS)抑制剂L-NAME,检测CCD大鼠背根神经节DRG内一氧化氮(NO)代谢产物亚硝酸盐(nitrite)含量变化,并观测热刺激缩爪反应潜伏期(PWL)的变化。结果:鞘内分别注射RR、TRPV4 AS ODN和L-NAME后,均能够显著降低CCD大鼠DRG内亚硝酸盐含量(P<0.05),CCD大鼠的热痛敏行为也能够显著改善(P<0.05)。结论:TRPV4及其下游信号分子NO参与介导CCD大鼠的热痛觉过敏。     

瞬时感受器电位离子通道4在大鼠背根神经节持续受压后异位放电中的作用

目的:利用在体神经纤维电生理技术记录大鼠背根神经节(DRG)持续受压(CCD)后的异位放电情况,明确瞬时感受器电位离子通道4(TRPV4)是否参与了CCD后受损DRG的异位放电。方法:共采用35只Wistar大鼠。制备大鼠DRG的CCD模型,分别于术前和术后测量损伤侧的机械痛阈和热辐射刺激缩爪反应潜伏期。利用在体神经纤维电生理技术分别记录正常组大鼠DRG及CCD组、CCD+钌红(RR)组、CCD+佛波醇(4α-PDD)组受损DRG神经元的异位放电情况。结果:持续压迫明显降低大鼠损伤侧的机械痛阈和热辐射刺激缩爪反应潜伏期(n=30,P0.05);CCD组可以记录到受损DRG神经元的异位放电,放电率约为67%,而正常组DRG的异位放电率约为4.5%;以TRP家族阻断剂钌红(RR)100μm孵育受损DRG,较CCD组受损DRG神经元异位放电的频率和波幅均明显下降(n=10,P0.05);以TRPV4特异性激动剂佛波醇(4α-PDD)10μm孵育受损DRG,较CCD组受损DRG异位放电频率和波幅均明显增加(n=10,P0.05)。结论:CCD后受损DRG可出现异位放电,TRPV4参与了CCD后受损DRG的异位放电。     

大鼠脊髓背角TRPV4在背根神经节持续受压致神经病理性疼痛中的作用

目的:探讨大鼠背根神经节(dorsal root ganglion,DRG)持续受压(chronic compression of right side dorsal root ganglion,CCD)后脊髓背角瞬时感受器电位离子通道4(TRPV4)基因及蛋白变化,明确脊髓背角TRPV4在CCD致神经病理性疼痛中的作用。方法:采用健康成年雄性Wistar大鼠,共36只,随机分为3组,分别为空白对照组、CCD手术组、CCD+钌红组。制备大鼠背根神经节持续受压模型,于术前1天、术后第7天、给药前及给药2h后,测量大鼠机械刺激缩爪反应阈值,观察机械痛阈的变化;利用RT-PCR及Western Blot技术检测各组大鼠手术侧脊髓背角TRPV4基因及蛋白表达的变化。结果:与空白对照组相比,术后第7天,CCD组大鼠术侧机械痛阈值明显下降(P0.001),同侧脊髓背角TRPV4基因及蛋白表达升高(P0.05);与给药前相比,给予钌红2h后,术侧机械痛阈值明显升高(P0.001),同侧脊髓背角TRPV4基因及蛋白表达下降(P0.05)。结论:CCD后大鼠术侧机械痛阈下降,脊髓背角TRPV4基因及蛋白表达升高;钌红可部分逆转CCD后痛觉过敏,部分降低脊髓背角TRPV4基因及蛋白表达。脊髓背角TRPV4参与CCD后大鼠神经病理性疼痛形成。     

低能量激光对大鼠背根神经节持续受压后痛觉敏感及TRPV4表达的影响

目的:观察低能量激光对大鼠背根神经节持续受压(chronic compression of the dorsal root ganglion,CCD)后痛觉敏感及脊髓背角内TRPV4表达及分布的影响。方法:选取健康雄性Wistar大鼠36只,分为空白组、CCD组、激光组各12只,各组均给予常规饲养。激光组在CCD后第4天经皮肤给予L4/L5脊髓节段处810nm低能量激光治疗7天,输出功率150mw,10min/次。并于术前,术后第4、7、11天,分别测量各组大鼠机械刺激缩爪反应阈值和热缩爪潜伏期,观察机械痛阈和热痛阈的变化。同时,利用Western Blot检测各组术后11天TRPV4在脊髓背角中蛋白表达的变化,利用免疫荧光技术检测各组术后11天TRPV4阳性细胞分布。结果:与空白组相比,CCD组在术后第4、7、11天,机械痛阈和热痛阈均降低(P<0.01),与CCD组相比,激光组在术后第7、11天时,机械痛阈和热痛阈明显增高(P<0.01)。与空白组相比较,CCD组大鼠脊髓背角中TRPV4表达显著上调(P<0.01),TRPV4阳性细胞数目升高;给予低能量激光治疗后,激光组脊髓背角TRPV4的蛋白表达较CCD组显著下调(P<0.01),TRPV4阳性细胞数目低于CCD组(P<0.01)。结论:低能量激光治疗可降低CCD后大鼠脊髓背角中TRPV4蛋白表达,减少阳性细胞数目,影响TRPV4对神经病理性疼痛的中枢敏化的作用,缓解CCD后所致的痛觉敏感。     

多节段背根节慢性压迫大鼠的机械触刺激和冷刺激诱发痛行为

目的:脊椎间盘突出、椎间孔狭窄、脊髓损伤以及肿瘤造成的背根节(dorsal root ganglion,DRG)及其邻近神经根的机械性压迫可能是引起腰背痛与坐骨神经痛的重要原因.临床上多节段神经根性痛比单一神经根性痛更常见,患者表现出多节段神经根压迫和多节段椎间孔狭窄.为此,本实验观察了多节段DRG慢性压迫大鼠的痛行为.方法:实验在本室创建的大鼠背根节慢性压迫(chronic compression of DRG,CCD)模型基础上采用单侧L3-5多节段DRG慢性压迫模型,应用von Frey细丝和丙酮分别检测机械触刺激诱发痛阚值和冷刺激诱发痛反应级别及反应百分数.结果:多节段DRG慢性压迫大鼠表现明显双侧机械触刺激诱发痛和冷刺激诱发痛行为,伴随明显延迟的对侧机械触刺激和冷刺激诱发痛的镜像痛行为.组织学观察显示多节段DRG慢性压迫同侧DRG内部及其神经根有明显炎症反应.结论:机械性压迫和炎症共同作用神经根和DRG导致多节段DRG慢性压迫大鼠明显的机械触刺激诱发痛和冷刺激诱发痛行为.     

大鼠背根神经节持续受压后脊髓背角TRPV4蛋白表达及分布的变化

目的:探讨大鼠背根神经节持续受压(chronic compression of the dorsal root ganglion,CCD)后瞬时感受器电位离子通道香草素亚族受体4(transient receptor potential vanilloid 4,TRPV4)在脊髓背角中的蛋白表达及分布规律。方法:选取清洁级雄性Wistar大鼠30只,按随机数字表法分组:空白对照组5只,手术组25只(术后4天、7天、14天、28天各5只;免疫荧光组5只)。制备大鼠背根神经节持续受压模型,于术后4天,7天,14天及28天,测量机械刺激缩爪反应痛阈,观察机械痛阈的变化;利用western blot技术检测空白对照组及术后4天,7天,14天及28天,手术侧及对侧脊髓背角TRPV4蛋白表达的变化;利用免疫荧光技术检测术后4天,手术侧与对侧脊髓背角TRPV4阳性细胞分布。结果:大鼠背根神经节持续受压后4—14天,机械痛阈值明显下降(P0.001);术后4—7天,手术侧脊髓背角TRPV4表达升高(P0.01),对侧无明显变化;手术侧脊髓背角内TRPV4阳性细胞数目高于对侧(P=0.0008)。结论:大鼠背根神经节持续受压后,机械痛阈下降的同时,手术侧脊髓背角内TRPV4蛋白表达上调及阳性数目增多,TRPV4可能参与CCD后病理性神经痛的中枢敏化机制。     

低频电针对脊神经结扎大鼠痛敏化的干预作用

目的观察低频电针对脊神经结扎神经病理痛大鼠模型痛敏化的干预作用,探讨其可能的外周痛敏化调节机制。方法建立大鼠L5脊神经结扎模型,电针足三里和昆仑穴,观察大鼠痛觉超敏反应,运用Western blotting 法检测L4、L5背根神经节(DRG)辣椒素受体(TRPV1)与P 物质(SP)水平,采用TRPV1 激动剂6'-IRTX进行验证。结果脊神经结扎大鼠出现明显的痛敏化反应,术侧L4 DRG TRPV1 和L5 DRG SP 水平升高(P<0.05);低频电针能减轻模型大鼠的痛敏反应,抑制TRPV1、SP 水平上升(P<0.05)。6'-IRTX腹腔注射能拮抗低频电针的抗痛敏化作用。结论低频电针可减轻痛敏化,发挥对神经病理痛的治疗作用,其机制可能与其有效调控DRG TRPV1 和SP有关。     

大鼠部分坐骨神经损伤模型中机械痛敏和冷痛敏的性别差异

目的 :检测大鼠神经部分损伤所致实验性神经病理性痛发生中的性别差异。方法:在成年大鼠中建立部分坐骨神经损伤模型,检测损伤后的机械性痛觉超敏和冷触诱发痛的发展,对比雌雄差异。结果:部分坐骨神经损伤可引起大鼠同侧后足对机械刺激和冷刺激的痛敏反应。虽然雌雄大鼠在机械痛敏程度上没有显著性差异,但雌性大鼠较雄性更早发生痛敏(雌鼠术后1天,雄鼠术后3天)且持续的时程更长(雌鼠术后84天,雄鼠术后49天)。雌性大鼠的冷痛敏比雄性在程度上有显著的增强(P<0.01)。结论:部分坐骨神经损伤引起的大鼠对机械刺激和冷刺激的痛敏现象存在显著的性别差异——雌性大鼠较雄性大鼠表现出更强的神经病理性痛样行为。     

Cav3.2通道对背根节慢性压迫痛大鼠脊髓CaMKⅡ表达的影响

目的:观察Cav3.2通道对背根节慢性压迫痛大鼠脊髓CaMKⅡ表达的影响,探讨Cav3.2-CaMKⅡ通路在神经病理性疼痛中的作用。方法:雄性SD大鼠60只,体重250±20 g,随机分为5组,每组12只(其中行为学实验8只,免疫印迹实验4只)。分别是正常对照组(C组);模型组(CCD组);生理盐水组(NS组);错义寡聚核苷酸组(MS-Cav3.2组);反义寡聚核苷酸组(AS-Cav3.2组)。分别于鞘内置管前(T1),鞘内置管后3 d(T2),鞘内给药后1 d(T3)、4 d(T4),CCD模型制备后5d(T5)、10 d(T6)、15 d(T7)检测大鼠机械缩腿阈值(mechanical withdrawal threshold,MWT)和热缩足潜伏期(thermal withdrawal latency,TWL)。并于CCD模型制备后5 d,各组取4只大鼠处死,取脊髓腰膨大,采用免疫印迹法检测Cav3.2及CaMKⅡ的表达。结果:与C组比较,CCD组大鼠在模型制备后第5 d、10 d、15 d时MWT及TWL均显著降低。鞘内注射NS和Cav3.2错义寡聚核苷酸对CCD大鼠MWT及TWL无影响,鞘内注射Cav3.2反义寡聚核苷酸可增加CCD大鼠MWT和TWL,减轻大鼠机械痛敏和热痛敏。C组大鼠脊髓Cav3.2和CaMKⅡ蛋白均有表达。大鼠在制备CCD模型后5 d Cav3.2及CaMKⅡ蛋白表达均显著增加。鞘内注射NS及Cav3.2错义寡聚核苷酸对Cav3.2及CaMKⅡ蛋白表达无影响,鞘内注射Cav3.2反义寡聚核苷酸则可显著抑制Cav3.2及CaMKⅡ蛋白的表达。结论:抑制脊髓Cav3.2通道的表达可降低慢性背根节压迫痛大鼠脊髓CaMKⅡ的表达。     

慢病毒载体介导的BDNF基因与电针对神经病理痛模型大鼠痛阈的影响

目的观察慢病毒载体介导的BDNF与电针对神经病理痛模型大鼠热辐射痛阈的影响差别,比较基因与电针的镇痛疗效,探讨BDNF对疼痛可能的作用机理。方法本研究采用坐骨神经结扎损伤的神经病理性疼痛大鼠CCI模型,运用荧光免疫标记法,观察慢病毒载体介导的BDNF与电针干预后大鼠不同时间点（3 d、7 d、21 d）BDNF在相应节段脊髓内表达的变化。另运用BME-410C热痛刺激仪记录大鼠术前（0 d）及术后1 d、3 d、5 d、7 d、9 d、14 d、21 d,各组的热辐射刺激缩足潜伏期痛阈的变化。结果大鼠热痛敏阈值的变化：各组大鼠CCI术前热痛敏阈值均无明显差异。模拟对照组大鼠各时点热痛敏阈值无明显差异。阴性对照组与空白对照组各时点相比无统计学差异。CCI术后一天各组热痛敏阈值低于模拟对照组（P〈0.05）。术后第5天,试验组、电针组热痛敏阈值上升,高于空白及阴性对照组（P〈0.05）,术后7天,实验组上升至最高点,试验组与电针组无明显差异（P〉0.05）。术后9天、14天、21天,实验组热痛敏阈值下降,术后7天、9天、14天电针组热痛敏阙值趋于稳定,均高于空白及阴性对照组（P〈0.05）,术后9天、14天实验组与电针组有明显差异（P〈0.05）,术后21天,试验组与电针组无明显差异（P〉0.05）。荧光免疫组化观察：空白对照组、阴性对照组及模拟对照组术后第3天、第7天、第21天时点大鼠L4/5段脊髓阳性神经元表达无明显变化（P〉0.05）,试验组三时点阳性神经元先增加后降低,术后第7天最高,与其他组相比阳性神经元表达明显升高（P〈0.05）,电针组阳性神经元有所增加,但与阴性对照组无明显差异（P〉0.05）。结论 BDNF参与了大鼠痛敏的过程。可能外源性BDNF表达有利于受损神经修复。     

Involvement of TRPV1 in Nociceptive Behavior in a Rat Model of Cancer Pain

To investigate the mechanisms underlying cancer pain, we developed a rat model of cancer pain by inoculating SCC-158 into the rat hind paw, resulting in squamous cell carcinoma, and determined the time course of thermal, mechanical sensitivity, and spontaneous nocifensive behavior in this model. In addition, pharmacological and immunohistochemical studies were performed to examine the role played by transient receptor potential vanilloid (TRPV)1 and TRPV2 expressed in the dorsal root ganglia. Inoculation of SCC-158 induced marked mechanical allodynia, thermal hyperalgesia, and signs of spontaneous nocifensive behavior, which were diminished by systemic morphine administration. Intraplantar administration of the TRPV1 antagonist capsazepine or TRP channels antagonist ruthenium red did not inhibit spontaneous nocifensive behavior at all. However, intraplantar administration of capsazepine or ruthenium red completely inhibited mechanical allodynia and thermal hyperalgesia produced by SCC-158 inoculation. Immunohistochemically, the number of TRPV1-positive, large-sized neurons increased, whereas there was no change in small-sized neurons in the dorsal root ganglia. Our results suggest that TRPV1 play an important role in the mechanical allodynia and thermal hyperalgesia caused by SCC-158 inoculation.

Perspective

We describe a cancer pain model that induced marked mechanical allodynia, thermal hyperalgesia, signs of spontaneous nocifensive behavior, and upregulation of TRPV1. Mechanical allodynia and thermal hyperalgesia were inhibited by TRP channel antagonists. The results suggest that TRPV1 plays an important role in the model of cancer pain.     

Peripheral inflammation induces up-regulation of TRPV2 expression in rat DRG

The transient receptor potential vanilloid subfamily member 2 (TRPV2) is a cation channel activated by temperatures above 52 degrees C. To analyze the contribution of TRPV2 to the development of inflammation-induced hyperalgesia, the expression of TRPV2 in primary sensory neurons was analyzed after intraplantar injection of complete Freund's adjuvant (CFA). Using specific antibodies, an increase in TRPV2-expressing neurons was identified after inflammation. TRPV2 expression is concentrated in a subset of medium-sized dorsal root ganglion neurons, independent of transient receptor potential vanilloid subfamily member 1 (TRPV1) expression. A similar distribution of TRPV2 was observed after inflammation. Intraplantar injection of nerve growth factor increased TRPV1 expression but not TRPV2, suggesting that induction of TRPV2 expression is driven by a mechanism distinct from that for TRPV1. Heat hyperalgesia assessment after chemical desensitization of TRPV1 by resiniferatoxin demonstrates a possible role for TRPV2 in inflammation at high temperatures (>56 degrees C). These results suggest that TRPV2 upregulation contributes to peripheral sensitization during inflammation and is responsible for pain hypersensitivity to noxious high temperature stimuli.     

Effect of paclitaxel on transient receptor potential vanilloid 1 in rat dorsal root ganglion

Peripheral neuropathy is a common adverse effect of paclitaxel treatment. To analyze the contribution of transient receptor potential vanilloid 1 (TRPV1) in the development of paclitaxel-induced thermal hyperalgesia, TRPV1 expression in the rat dorsal root ganglion (DRG) was analyzed after paclitaxel treatment. Behavioral assessment using the tail-flick test showed that intraperitoneal administration of 2 and 4 mg/kg paclitaxel induced thermal hyperalgesia after days 7, 14, and 21. Paclitaxel-induced thermal hyperalgesia after day 14 was significantly inhibited by the TRP antagonist ruthenium red (3 mg/kg, s.c.) and the TRPV1 antagonist capsazepine (30 mg/kg, s.c.). Paclitaxel (2 and 4 mg/kg) treatment increased the expression of TRPV1 mRNA and protein in DRG neurons. Immunohistochemistry showed that paclitaxel (4 mg/kg) treatment increased TRPV1 protein expression in small and medium DRG neurons 14 days after treatment. Antibody double labeling revealed that isolectin B4-positive small DRG neurons co-expressed TRPV1. TRPV1 immunostaining was up-regulated in paw skin day 14 after paclitaxel treatment. Moreover, in situ hybridization histochemistry revealed that most of the TRPV1 mRNA-labeled neurons in the DRG were small or medium in size. These results suggest that paclitaxel treatment increases TRPV1 expression in DRG neurons and may contribute to functional peripheral neuropathic pain.     

AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide], a novel vanilloid receptor 1 (TRPV1) antagonist with antihyperalgesic properties

The vanilloid receptor 1 (VR1 or TRPV1) is a membrane-bound, nonselective cation channel expressed by peripheral sensory neurons. TRPV1 antagonists produce antihyperalgesic effects in animal models of inflammatory and neuropathic pain. Here, we describe the in vitro and in vivo pharmacology of a novel TRPV1 antagonist, AMG 9810, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide. AMG 9810 is a competitive antagonist of capsaicin activation (IC50 value for human TRPV1, 24.5 +/- 15.7 nM; rat TRPV1, 85.6 +/- 39.4 nM) and blocks all known modes of TRPV1 activation, including protons (IC50 value for rat TRPV1, 294 +/- 192 nM; human TRPV1, 92.7 +/- 72.8 nM), heat (IC50 value for rat TRPV1, 21 +/- 17 nM; human TRPV1, 15.8 +/- 10.8 nM), and endogenous ligands, such as anandamide, N-arachidonyl dopamine, and oleoyldopamine. AMG 9810 blocks capsaicin-evoked depolarization and calcitonin gene-related peptide release in cultures of rat dorsal root ganglion primary neurons. Screening of AMG 9810 against a panel of G protein-coupled receptors and ion channels indicated selectivity toward TRPV1. In vivo, AMG 9810 is effective at preventing capsaicin-induced eye wiping in a dose-dependent manner, and it reverses thermal and mechanical hyperalgesia in a model of inflammatory pain induced by intraplantar injection of complete Freund's adjuvant. At effective doses, AMG 9810 did not show any significant effects on motor function, as measured by open field locomotor activity and motor coordination tests. AMG 9810 is the first cinnamide TRPV1 antagonist reported to block capsaicin-induced eye wiping behavior and reverse hyperalgesia in an animal model of inflammatory pain.     

Analgesic Effects of Transcutaneous Ultrasound Nerve Stimulation in a Rat Model of Oxaliplatin-Induced Mechanical Hyperalgesia and Cold Allodynia

This study investigated the effects and underlying mechanisms of therapeutic ultrasound (TUS) in a rat model of oxaliplatin-induced peripheral neuropathy. Animals received a total of eight injections with oxaliplatin (4 mg/kg), administered at 3-d intervals. TUS intervention (1 MHz, 0.5 W/cm²) started on the fifth oxaliplatin administration and continued for 10 consecutive d. Sensory behavioral examinations, protein levels of transient receptor potential channels (TRPM8 and TRPV1) in dorsal root ganglia (DRG) and substance P (SP) in spinal dorsal horn were examined. Results indicated that TUS can reduce mechanical and cold hyper-responsive behaviors caused by repeated administration of oxaliplatin. Oxaliplatin-related increases in protein levels of TRPM8 in DRG and SP in the dorsal horn were also reduced after TUS. Taken together, the results revealed beneficial effects of TUS on oxaliplatin-induced mechanical hyperalgesia and cold allodynia and suggested involvement of TUS biochemicals in suppressing TRPM8 in DRG and SP in spinal cords.     

Sustained inhibition of neurotransmitter release from nontransient receptor potential vanilloid type 1-expressing primary afferents by mu-opioid receptor activation-enkephalin in the spinal cord

Removing transient receptor potential vanilloid type 1 (TRPV1)-expressing primary afferent neurons reduces presynaptic mu-opioid receptors but potentiates opioid analgesia. However, the sites and underlying cellular mechanisms for this paradoxical effect remain uncertain. In this study, we determined the presynaptic and postsynaptic effects of the mu-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO) using whole-cell patch-clamp recordings of lamina II neurons in rat spinal cord slices. Treatment with the ultrapotent TRPV1 agonist resiniferotoxin (RTX) eliminated TRPV1-expressing dorsal root ganglion neurons and their central terminals in the spinal dorsal horn and significantly reduced the basal amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from primary afferents. Although RTX treatment did not significantly alter the concentration-response effect of DAMGO on evoked monosynaptic and polysynaptic EPSCs, it causes a profound long-lasting inhibitory effect of DAMGO on evoked EPSCs. Subsequent naloxone treatment did not reverse the prolonged inhibitory effect of DAMGO on evoked EPSCs. Furthermore, brief application of DAMGO produced a sustained inhibition of miniature EPSCs in RTX-treated rats. However, the concentration response and the duration of the effects of DAMGO on G protein-coupled inwardly rectifying K+ currents in lamina II neurons were not significantly different between vehicle- and RTX-treated groups. These data suggest that stimulation of mu-opioid receptors on non-TRPV1 afferent terminals causes extended inhibition of neurotransmitter release to spinal dorsal horn neurons. The differential effect of mu-opioid receptor agonists on different phenotypes of primary afferents provides a cellular basis to explain why the analgesic action of opioids on mechanonociception is prolonged when TRPV1-expressing primary afferents are removed.     

A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel and selective transient receptor potential type V1 receptor antagonist, blocks channel activation by vanilloids, heat, and acid

The vanilloid receptor transient receptor potential type V1 (TRPV1) integrates responses to multiple stimuli, such as capsaicin, acid, heat, and endovanilloids and plays an important role in the transmission of inflammatory pain. Here, we report the identification and in vitro characterization of A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel, potent, and selective TRPV1 antagonist. A-425619 was found to potently block capsaicin-evoked increases in intracellular calcium concentrations in HEK293 cells expressing recombinant human TRPV1 receptors (IC50 = 5 nM). A-425619 showed similar potency (IC50 = 3-4 nM) to block TRPV1 receptor activation by anandamide and N-arachidonoyl-dopamine. Electrophysiological experiments showed that A-425619 also potently blocked the activation of native TRPV1 channels in rat dorsal root ganglion neurons (IC50 = 9 nM). When compared with other known TRPV1 antagonists, A-425619 exhibited superior potency in blocking both naive and phorbol ester-sensitized TRPV1 receptors. Like capsazepine, A-425619 demonstrated competitive antagonism (pA2 = 2.5 nM) of capsaicin-evoked calcium flux. Moreover, A-425619 was 25- to 50-fold more potent than capsazepine in blocking TRPV1 activation. A-425619 showed no significant interaction with a wide range of receptors, enzymes, and ion channels, indicating a high degree of selectivity for TRPV1 receptors. These data show that A-425619 is a structurally novel, potent, and selective TRPV1 antagonist.     

(R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102) blocks polymodal activation of transient receptor potential vanilloid 1 receptors in vitro and heat-evoked firing of spinal dorsal horn neurons in vivo

The transient receptor potential vanilloid (TRPV) 1 receptor, a nonselective cation channel expressed on peripheral sensory neurons and in the central nervous system, plays a key role in pain. TRPV1 receptor antagonism is a promising approach for pain management. In this report, we describe the pharmacological and functional characteristics of a structurally novel TRPV1 antagonist, (R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102), which has entered clinical trials. At the recombinant human TRPV1 receptor ABT-102 potently (IC(50) = 5-7 nM) inhibits agonist (capsaicin, N-arachidonyl dopamine, anandamide, and proton)-evoked increases in intracellular Ca(2+) levels. ABT-102 also potently (IC(50) = 1-16 nM) inhibits capsaicin-evoked currents in rat dorsal root ganglion (DRG) neurons and currents evoked through activation of recombinant rat TRPV1 currents by capsaicin, protons, or heat. ABT-102 is a competitive antagonist (pA(2) = 8.344) of capsaicin-evoked increased intracellular Ca(2+) and shows high selectivity for blocking TRPV1 receptors over other TRP receptors and a range of other receptors, ion channels, and transporters. In functional studies, ABT-102 blocks capsaicin-evoked calcitonin gene-related peptide release from rat DRG neurons. Intraplantar administration of ABT-102 blocks heat-evoked firing of wide dynamic range and nociceptive-specific neurons in the spinal cord dorsal horn of the rat. This effect is enhanced in a rat model of inflammatory pain induced by administration of complete Freund's adjuvant. Therefore, ABT-102 potently blocks multiple modes of TRPV1 receptor activation and effectively attenuates downstream consequences of receptor activity. ABT-102 is a novel and selective TRPV1 antagonist with pharmacological and functional properties that support its advancement into clinical studies.