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Anesth Pain Med 2014; 9: 93-97 실험연구 신경병증통증쥐척수강내로투여한 Tianeptine 의항이질통효과 * 전남대학교의과대학마취통증의학교실, 전남대학교창의적의과학인력사업단림해 *, ㆍ이형곤 * ㆍ윤명하 *, Antiallodynic effects of intrathecal tianeptine in a neuropathic pain rat Hai Lin*,, Hyung Gon Lee*, and Myung Ha Yoon*, *Department of Anesthesiology and Pain Medicine, Medical School, Center for Creative Biomedical Scientists at Chonnam National University, Gwangju, Korea Background: Tianeptine is an antidepressant drug which is used for treating depression. Interestingly, the tianeptine has shown antinociceptive effects within a variety of nociceptions. The aim of this study is to investigate the antiallodynic effects of tianeptine in neuropathic pain rats and also determine the involvements of serotonergic, alpha-2 adrenergic and adenosine receptors at the spinal level. Methods: Neuropathic pain was induced by ligation of left lumbar at 5th and 6th spinal nerves in male Sprague-Dawley rats. PE-10 catheters were placed into the thoracolumbar subarachnoid space for drug injections. Mechanical allodynia was evaluated by measuring the withdrawal threshold to von Frey filament when applying on the plantar surface of rats. The effects of intrathecal tianeptine were observed at 15, 30, 60, 90, 120, 150, 180 minutes after delivery. Antagonists for serotonergic (dihydroergocristine), alpha-2 adrenergic (yohimbine) and adenosine (CGS 15943) receptors were intrathecally administered 10 minutes prior to tianeptine in order to evaluate the involvement of both receptors. Results: Intrathecal tianeptine increased dose-dependently at the withdrawal threshold in the ligated paw. Pretreatment with intrathecal dihydroergocristine, yohimbine and CGS 15943 antagonized the antiallodynic effects of tianeptine. Conclusions: These results suggested that intrathecal tianeptine attenuates the spinal nerve ligation induced tactile allodynia. Received: September 6, 2013. Revised: December 26, 2013. Accepted: January 22, 2014. Corresponding author: Myung Ha Yoon, M.D., Ph.D., Department of Anesthesiology and Pain Medicine, Medical School, Chonnam National University, 8, Hak-dong, Dong-gu, Gwangju 501-757, Korea. Tel: 82-62-220-6893, Fax: 82-62-232-6294, E-mail: mhyoon@chonnam.ac.kr It was presented The 51th Annual Scientific Meeting of the Korean Pain Society, November 2010, Grandhilton Hotel, Seoul, Korea Serotonergic, alpha-2 adrenergic and adenosine receptors are all involved in the antiallodynic effects of tianeptine at the spinal level. (Anesth Pain Med 2014; 9: 93-97) Key Words: Adenosine receptor, Alpha-2 adrenergic receptor, Antiallodynia, Neuropathic pain, Serotonin receptor, Tianeptine. 서 신경병증통증은말초및중추신경손상후에몸감각 (somatosensory) 체계에병을일으켜발생하며특징적증상으로는통각과민 ( 유해자극에대한반응이더강화되어서나타나는통증반응 ) 과이질통 ( 무해한자극에도유발되는통증반응 ) 이있다 [1]. 이러한신경병증통증은임상적으로치료하기도쉽지않고환자도매우힘들어하며시간이지나면서환자의정상적인생활을힘들게하고심지어는심리적혹은정신적부작용까지도일으켜삶의질을떨어뜨려만성통증으로진행하게된다. 아울러흔히널리쓰이는일반진통제는신경병증통증에대한효과가미미하고현재널리쓰이는약물요법의효과도신경병증통증환자의 50% 에서는효과가충분하지않다 [2]. 따라서이러한한계점을극복하기위한새로운약물의개발은필수적이다. 최근척수수준에서만성통증을조절하는다양한약물학적전략에대한연구가활발하게진행되고있으며많은발전을이루고있다 [3]. 항우울제는항우울효과를나타내는용량보다도더적은용량에서항우울효과와는별도로진통효과를보이며 [4] 이러한진통작용은세로토닌과노르에피네프린재흡수를억제하여나타나며 [5] 아데노신체계도항우울제작용에관여한다고한다 [6]. Tianeptine은항우울제로서삼환계항우울제와구조가유사하나신경화학적특성은다르다 [7]. 이전동물실험에의하면 tianeptine은급성, 염증성및내장성통증을억제하였으나 [8-10] 신경병증통증에대한효과는알려진바가없다. 한편, 세로토닌, 아드레날린및아데노신은통증과같은유해한자극의전달을조절하는중요한역할을하면주요작용점은척수로알려져있다 [11-13]. 이전보고에의하면 론 93

94 Anesth Pain Med Vol. 9, No. 2, 2014 척수강내로투여한 tianeptine은포르말린유발통증을억제하였으며이는세로토닌성수용체길항제와 alpha-2 아드레날린성수용체길항제에의해역전되었다 [8]. 이는 tianeptine 의효과가이러한수용체와관련이있음을시사한다. 반면, tianeptine의작용에대한아데노신수용체에의역할에대해서는알려진바가없다. 따라서본연구에서는쥐의척수신경결찰로유발된신경병증통증에대한척수강 tianeptine의효과를관찰하고그작용에대한세로토닌성, alpha-2 아드레날린성및아데노신수용체역할을척수수준에서규명하고자하였다. 대상및방법실험동물의과대학동물윤리위원회로부터실험계획에대한승인을얻은후규정에따라모든실험을진행하였다. 수컷 Sprague-Dawley (100 200 g) 쥐를대상으로실험하였으며사육실은항온 (22 23 o C) 상태로유지하였고모든쥐는물과먹이를자유롭게섭취할수있도록하였다. 통증모형통증모형으로신경병증통증모형을이용하였다 [14]. 수컷 Sprague-Dawley (100 200 g) 쥐를대상으로신경병증통증유발을위한수술을시행하였다. Sevoflurane 마취하에서쥐를복와위로고정하고제 4 요추부에서제 2 천추부위까지피부절개하고좌측척추옆근육을견인한후좌측제 5, 6 요추부의척추신경을분리하여 6-0 실크로단단히결찰하였다. 1주일동안매일기계적자극 (von Frey filaments) 에대한역치를측정하여 4 g 이하인쥐만을대상으로신경병증통증모형을위한수술후 7일째에척수강내카테터를거치하였다. 척수강카테터삽입술척수강내카테터는 Yaksh와 Rudy의방법으로거치하였다 [15]. Sevoflurane 마취하에서쥐를뇌정위고정장치에고정하고두개골위에서정중선을따라양귀 2 cm 하방까지절개하고근육과근막을견인한후환추후두막을찾은다음이경막을 23 게이지바늘끝으로절개한후 polyethylene (PE-10) 카테터를 8 cm 하방까지삽입하였다. 카테터외부는피하를통하여두개골상부에서고정한다음 28 게이지철사로막아놓았다. 절개부위를 3-0 실크로봉합하고마취에서각성시켰다. 카테터삽입후신경학적손상을보인쥐는실험에서제외하고흡입마취제과다투여로바로사망시켰으며정상소견을보이는쥐는각각의케이지에넣어수술 5일후에본실험을시행하였다. 약물및투여본연구의실험약물은 tianeptine (provided by JEIL Pharm. Co., Ltd., Seoul, Korea), dihydroergocristine (Sigma-Aldrich Co., St. Louis, MO, USA), yohimbine (Sigma-Aldrich) 및 CGS 15943 (Sigma-Aldrich) 이었다. 약물은증류수에용해시켰으며척수강내로투여하기위해서는투여량을 10 μl로하여수동식기어장치주사기에카테터를연결하여척수강내로주입하고카테터내의사강용적을고려하여실험약물주입후생리식염수 10 μl를추가로주입하였다. 실험방법모든실험은처치된약물에대해모르는관찰자에의해시행되었다. 실험당일쥐를투명한철사상자 (21 27 15 cm) 로옮기고 15 20분정도의적응기간이지나면실험을시행하였다. 이질통역치측정 : 통증정도는기계적자극에의한회피반응값을측정하여평가하였다. 기계적자극은 von Frey filaments를이용하였다. 철사상자바닥의구멍을통하여 von Frey filaments로쥐의발바닥에자극을가한후 updown 법을이용하여회피반응을측정하였다 [16]. 즉, 8개의 filament (0.4, 0.7, 1.2, 2.0, 3.6, 5.5, 8.5, and 15 g) 를이용하여 filament가약간휘어질정도로 6초씩자극을가했을때급작스런발의회피반응을보이거나발을핥을경우양성반응으로간주하였다. 양성반응을보일경우다음자극시에는더가벼운 filament하며반대로음성일경우에는더무거운 filament로자극을하였다. 이러한일련의과정을거쳐회피반응을보이는최하 filament 무게를회피반응값으로규정하였다. 15 g의자극에도반응을보이지않는경우, 15 g을 cut-off값으로하였다. Tianeptine 효과 : 신경결찰후 12일에실험약물인 tianeptine (30 μg, n = 6; 100 μg, n = 6; 300 μg, n = 6) 과생리식염수 (n = 5) 를척수강내로투여하고 15, 30, 60, 90, 120, 150, 180분에 von Frey filaments에대한회피반응을측정하였다. 실험약물투여직전미리측정하여얻은회피반응값을기준값으로하였다. Tianeptine 효과에대한세로토닌과아데노신수용체의역할 : Tianeptine의작용에대한세로토닌성, alpha-2아드레날린성및아데노신수용체의역할을알아보기위하여세수용체의길항제인 dihydroergocristine (3 μg, n = 5), yohimbine (10 μg, n = 5) 과 CGS 15943 (0.03 μg, n = 5) 을 tianeptine (300 μg, n = 5) 투여 10분전에척수강내로미리투여하고 tianeptine 효과의길항여부를관찰하였다. 길항제의용량은예비실험을통하여회피반응에영향이없으면서또한운동장애도유발하지않는최대용량으로하였다. 행동변화관찰 : Tianeptine (300 μg), dihydroergocristine

림해외 2 인 :Tianeptine 과항이질통작용 95 Fig. 1. Time effect (A) and dose-response (B) curves of intrathecal tianeptine for hindpaw withdrawal response to von Frey filaments after nerve ligation. Data are presented as the withdrawal threshold or the percent of maximal possible effect (%MPE). Each point on the graph represents median (interquartile) of 5 6 rats. B = baseline withdrawal threshold measured immediately before the drug injection. Intrathecal tianeptine produced a dose-dependent increase in the withdrawal threshold in the ligated paw. *P < 0.05, P < 0.01, compared with vehicle. (3 μg), yohimbine (10 μg) 과 CGS 15943 (0.03 μg) 에의한행동변화를알아보기위하여다른쥐를이용하여 (n = 10) 약물을투여한후이개 (pinna) 반사 (PE-10 카테터로외이도를자극하면귀를움직이는반사 ), 각막 (corneal) 반사 (PE-10 카테터로각막을자극하면눈을움직이는반사 ) 를관찰하고운동기능의평가를위해위치-보행 (placing-stepping) 반사 ( 실험쥐를책상의모서리에두면발로꽉잡고올라서려는반응 ) 및바로잡기 (righting) 반사 ( 실험쥐를책상바닥에뒤짚어두면바른자세를취하려는반응 ) 를관찰하였다 [17]. 모든반사는존재유무로판단하였다. 자료및통계분석 모든측정값은중앙값 ( 사분위수 ) 로표시하였다. 시간반응자료는회피반응의무게 (g) 로하였다. 용량반응자료는아래공식에따라최대가능효과 (maximal possible effect) 백분율 (%MPE) 로하고 ED 50 을구하였다. 약물투여후회피반응무게 대조군회피반응무게 대조군회피반응무게 용량반응자료분석은 Friedman test로하였고사후검증으로 Mann-Whitney test를이용하였다. Tianeptine 효과에대한길항자료는 Mann-Whitney test를이용하여분석하였다. P값이 0.05 미만일때를통계적으로유의하다고판정하였다. 결 척수강내로투여한 tianeptine은결찰술을받은쥐의발바닥회피반응역치를용량에비례하여증가시켰으며약물 과 투여후 15분에최고에도달했다 (Fig. 1). 아울러항이질통효과는용량의존적형태를보였다 (Fig. 1). 최대용량인 300 μg에서의 MPE는 86% 이었다. 척수강내로투여한세로토닌성, alpha-2 아드레날린성및아데노신수용체길항제인 dihydroergocristine, yohimbine 및 CGS 15943는척수강 tianeptine의항이질통효과를각각길항시켰다. 세길항제자체는모두회피반응에영향이없었다 (Fig. 2). 각막반사, 이개반사, 위치-보행반사및바로잡기반사는본연구에서투여한척수강 tianeptine, dihydroergocristine, yohimbine과 CGS 15943의최대용량에서도모두정상적으로존재하였다. 고찰최근우리나라는급격한고령화로만성통증질환의발병률이증가하는추세이다. 따라서대상포진후신경통이나당뇨병성신경통등말초성신경병증통증환자가증가하고있다. 그러나침해수용기통증이나염증성통증은아편양제제나비스테로이드성항염증제등에의해잘조절되나신경병증통증은이러한약물에큰효과가없으며또한선택적약물도없는실정이어서만족할만한수준의치료가제공되지못하고있다. 따라서이러한통증을극복하기위한연구가절실한실정이고최근그러한노력이활발하게증가하고있다. 본연구에서도항우울제를이용하여신경병증통증을극복하기위한일환으로작용기전이잘알려져있지않은 tianeptine을사용하여그효과와기전을규명하였다. 본연구에서는척수강내로투여한 tianeptine은척추신경

96 Anesth Pain Med Vol. 9, No. 2, 2014 Fig. 2. Antagonistic effects of intrathecal dihydroergocristine (HEC, 3 μg), yohimbine (10 μg) and CGS 15943 (0.03 μg) on the antiallodynic effect of intrathecal tianeptine (100 μg) in spinal nerve ligation induced neuropathic pain. HEC, yohimbine and CGS 15943 were given 10 min before tianeptine administration. Data are presented as percentages of maximal possible effect (%MPE). Each bar represents median (interquartile) of 5 rats. B = baseline withdrawal threshold measured immediately before the drug injection. HEC, yohimbine and CGS 15943 reversed the antiallodynic effect of tianeptine. *P < 0.05, P < 0.01, compared with tianeptine. 결찰술에의해유발된신경병증통증을유의하게억제하였다. 이는 tianeptine이척수수준에서신경병증통증을억제할수있다는것을시사한다. Tianeptine은 dibenzothiazepine 유도체항우울제로우울증이나이를동반한불안중울효과적으로감소시킨다고한다 [18]. 또한 tianeptine은기존삼환계항우울제에비해진정작용과심혈관계부작용이더적다고한다 [19]. 기전적으로 tianeptine은기존삼환계항우울제가세로토닌흡수를억제하는것과는대조적으로뇌에서세로토닌흡수를증가시키는것으로알려져있다 [20]. 이러한 tianeptine 작용의차이점에대한명확한기전은분명하지않지만, 어쨌든이는 tianeptine이세로토닌과관련하여작용을나타낸다는것을시사한다. 한편통증에대한 tianeptine의효과에대한자료는그리많지않다. 이전보고에의하면복강내로투여한 tianeptine은 tail-flick과 hot-plate 시험에서진통작용을나타냈다 [9]. 또한, 척수강내로투여한 tianeptine은포르말린유발통증을억제하였고정맥내로투여한 tianeptine은대장확장에의해유도된내장통을현저히감소시켰다 [8,10]. 그러나신경병증통증에대한 tianeptine의효과와작용기전은명백히밝혀진바가없다. 본연구결과 tianeptine의진통작용은척수강내로투여한세로토닌성, alpha-2아드레날린성및아데노신수용체길항제에의해역전되었다. 세로토닌은통증조절에관여하며 7 종류의수용체아형이있다 [11,21]. 이전보고에의하면척 수강내로투여한 tianeptine은포르말린주입으로유발된염증성통증을억제하였으며그진통작용은척수강으로투여한세로토닌길항제에의해역전되었다 [9]. Alpha-2 아드레날린성수용체길항제도포르말린시험에서 tianeptine의진통작용을길항시켰으며 3종류의아형이있다 [8,12]. 한편, 아데노신은내인성퓨린계물질로써통증조절에관여하며 4종류의수용체아형이있다 [13]. 신경병증통증에대한 tianeptine의효과가아데노신수용체를중재하여나타난다는결과는본연구가최초이다. 본실험결과를종합하면 tianeptine은척수신경결찰후발생하는신경병증통증을억제하였으며이러한항이질통효과는척수에서세로토닌성, alpha-2 아드레날린성및아데노신수용체를중재하여나타난다는것을의미한다. 그러나각수용체아형중에서어떤수용체가 tianeptine의진통작용에더연관되어있는지는본실험만으로는알수가없었고이부분은향후연구에서추가적으로더규명되어야할것으로생각된다. 아울러활성화된세가지수용체가어떤회로를통하여항이질통효과를나타내는지에대한연구도필요하다. 신경병증통증조절은환자들의통증을감소시키며정신적안정감을제공하게된다. 따라서효과적인통증조절은환자의만족도를증진시키고환자의삶의질을향상시키는것이다. 특히통증자극전달조절에관여하는물질들이많아여러종류의약제들이사용되고있으며동시에연구중에있다. 따라서본연구도신경병증조절을위한임상적사용이가능한약제의개발에도이바지할것으로사료된다. 결론적으로척수강내로투여한 tianeptine은척수신경결찰에의해유발된신경병증통증을효과적으로억제하였고이러한작용은척수의세로토닌성, alpha-2 아드레날린성및아데노신수용체를매개하는것으로생각된다. 감사의글 This study was financially supported by Chonnam National University, 2011. 참고문헌 1. Treede RD, Jensen TS, Campbell JN, Cruccu G, Dostrovsky JO, Griffin JW, et al. Neuropathic pain: redefinition and a grading system for clinical and research purposes. Neurology 2008; 70: 1630-5. 2. Wallace JM. Update on pharmacotherapy guidelines for treatment of neuropathic pain. Curr Pain Headache Rep 2007; 11: 208-14. 3. Zeilhofer HU, Benke D, Yevenes GE. Chronic pain states: pharmacological strategies to restore diminished inhibitory spinal pain control. Annu Rev Pharmacol Toxicol 2012; 52: 111-33.

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