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1 대한배뇨장애요실금학회지 J Korean Continence Soc 29;13:152-8 원저 과민성방광모델로서자연발생고혈압쥐에서비마취하요역동학검사로관찰한항콜린성제제인 tolterodine 의배뇨압력및용적에대한경구및주사효과 인하대학교의과대학비뇨기과학교실, 1 SK 생명과학연구센터 이소영 1 진롱후 권용현 장진혁 강용진 윤상민 이택 The Effect of tolterodine Via Oral and Intravenous Administrations on Voiding in Awake Spontaneously Hypertensive Rats as an Overactive Bladder Model So-young Lee 1, Long-Hu Jin, Yong-Hyun Kwon, Jin-Hyuk Jang, Yong-Jin Kang, Sang-Min Yoon, Tack Lee From the Department of Urology, Inha University Medical College, Incheon, Korea, 1 SK Chemicals Life Science R&D Center Pharmacology Team Purpose: We investigated the effect of oral or intravenous tolterodine on cystometric parameters in awake spontaneously hypertensive rats (SHRs) as a model of overactive bladder (OAB). The aim of our study was to observe the experimental conditions required to reproduce the clinical pharmacological effects of tolterodine, as seen in humans, to decrease bladder pressure or increase bladder capacity. Materials and methods: We studied the effects of the most widely used antimuscarinic drug, tolterodine, on cystometric parameters via two different administrations (oral and intravenous) in awake SHRs. Results: Oral administration of tolterodine 1 mg/kg -1 body weight in awake rats did not change any cystometric parameters significantly. Intravenous administration of tolterodine.3 mg/kg -1 body weight significantly decreased basal pressure (BP) and micturition pressure (MP), but showed no effect on micturition interval (MI) or bladder capacity (BC). Conclusion: Despite a high dose of tolterodine via an oral or an intravenous route, a decrease in BP or MP was the only effect on cystometrographic parameters in awake rats, whereas MI and BC were not significantly affected. Therefore, it is difficult to reproduce in awake rats as an acute response the cystometric increase in the MI that is observed in humans after chronic administration of antimuscarinic agents. (J Korean Continence Soc 29;13:152-8) Key Words: Tolterodine; cystometry; Spontaneous hypertensive rat 1) 접수일자 : 29 년 12 월 21 일, 수정일자 : 29 년 11 월 23 일, 채택일자 : 29 년 12 월 24 일 Address for correspondence: Tack Lee Department of Urology, Inha University College of Medicine by BK 21 project, Incheon, Korea Tel: , Fax: , lt11@inha.ac.kr 152 대한배뇨장애요실금학회지

2 비마취하자연발생고혈압쥐에서 tolterodine 의경구및주사효과 서론과민성방광은유병률이높고, 고령에서의발생률이증가하는질환으로국민건강적측면에서도매우중요한질환이며 [1,2], 이런이유로국제적인약물산업측면에서도중요한주제가되고있다. 부교감신경계의자극에의해방광수축이나타난다는것이밝혀지면서, 과민성방광의일차치료약물로항콜린성제제들이사용되고있다 [3]. 이러한항콜린성제제들은골반신경에서분비된 acetylcholine과경쟁적으로평활근내무스카린수용체에결합함으로써, 저장기에갑자기나타나는비억제성배뇨근수축을억제하는것으로알려져있다. 그러나, 배뇨시에도 acetylcholine이작용하여방광의수축이일어나기때문에이시기에도항콜린성제제의경쟁적억제작용의가능성이제시되고있다 [4-5]. 항콜린성제제들에대한기존연구들은 [2-4], 항콜린성제제가동물의 in vitro 실험이나마취하요역동학검사에서배뇨근의수축력을억제하여배뇨근과활동성을감소시키며, 저장기의방광내압을감소시켜배뇨간격을늘리고, 배뇨시배뇨압력을감소시킨다고보고하고있고, 사람에서도유사한효과가확인되었다. 그러나항콜린성제제의효과가절대적이지않고적지않은부작용이발생하므로지속적으로과민성방광에대한새로운치료제가개발되고있다. 항콜린성제제를연구하기위해비마취하요역동학검사를시행하는것에대해서는아직많은논란이있다 [2,3,6,7]. 이러한논란의이유는사람에서효과가있다고밝혀진항콜린성제제가비마취하동물에서효과를보이지않는경우가있어연구중인새로운약제에대한효과판정이어려울수있고, 또한사람을대상으로할수없는부작용등의발생여부는비마취하동물에서도알수없기때문이다. 자연발생고혈압쥐 (spontaneous hypertensive rat: SHR) 는원래사람의고혈압에대한동물모델로개발되었지만, 최근에는과민성방광모델로사용되어지고있으며이들은빈뇨와함께요역동학검사에서배뇨근과활동성등을보여, 객관적으로공인된사람과민성방광에대한동물모델로여겨지고있다 [8]. 이에저자들은 SHR을이용한과민성방광모델에서항콜린성제제인 tolterodine을경구와정맥주사로주입하여비마취하배뇨시요역동학적압력및용적변수들의변화와복압에미치는영향을알아보고자하였다. 대상및방법 1) 실험동물체중 275 3g인 12주령의수컷 SHR (Orient Bio Inc., GyeongGi, Korea) 1마리를대상으로하였다. 실험계획안은본학교의동물윤리위원회에서승인을받았으며, 낮, 밤을 12시간씩되게조절하였고, 음식과물은규정된프로토콜에따라자유롭게먹을수있도록하였다. 2) 실험군모든 SHR에서 3 5회의배뇨를시킨후요역동학검사를시행하여요역동학검사의측정변수에대한대조값을구한뒤, 5마리에는경구로 tolterodine 1 mg/kg 을투여하였고, 다른 5마리에는정맥주사로 tolterodine.3 mg/kg을투여하였다. 약물투여후각군에서 3-5 회의배뇨를시킨후요역동학검사를다시시행하여요역동학검사의변수들을측정하였다. 경구투여군에서는약물투여한시간후요역동학검사를시행하였고, 주사군에서는약물투여직후요역동학검사를시행하였다. SHR은기존다른연구에서 Wistar rat에비해배뇨압이높고배뇨간격이줄어있는과민성방광모델로증명되어있어 [8], 본연구에서는 Wistar rat과의비교는시행하지않았고, SHR에서의약물주입전, 후를비교하여약물의배뇨변수에대한효과를관찰하였다. 3) 방광내관및복압측정관삽입비마취하요역동학검사시행 3일전에 Xylazine (Rompun, 바이엘코리아, 한국 15 mg/kg) 과 ketamine (Ketamine; 유한양행, 서울, 한국 75 mg/kg) 을복강내주입하여마취를시행한뒤, 하복부중앙절개를통하 Vol. 13, No. 2,

3 이소영 진롱후 권용현 장진혁 강용진 윤상민 이택 여, cuff가달린 polyethylene catheter (PE-5, Becton Dickinson, Parsippany, USA) 를방광꼭대기 (bladder dome) 에주머니끈봉합법 (purse-string suture) 으로고정하였다. 또한 polyethylene catheter (PE-1, Becton Dickinson, Parsippany, USA) 를 femoral vein에삽입후고정하였다. 상기 catheter들을피부아래쪽으로통과시킨후등쪽의피부에고정하였으며끝부분은불로지져막아놓았다. 4) 방광내압및배뇨량측정방광내관과복압측정관삽입 3일후신진대사우리 (metabolic cage) 에마취하지않은자유로운상태에서, T자관 (T-tube) 을이용하여방광내관을현미주사펌프 (PHD22/2 pump; Harvard Apparatus) 와압력측정계 (Reaserch Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, USA) 에연결한뒤, 1 ml/hour -1 의속도로실온의생리식염수를주입하여방광내압을측정하였다. 요채집컵 (fluid collector) 을 force displacement transducer (Reaserch Grade Isometric Transducer; Harvard Apparatus) 에연결하여이컵에모인소변양으로배뇨량을측정하였다. 방광내압과복압, 배뇨량의자료수집분석은 MP15 data acquisition system (BIOPAC systems, Goleta, USA) 과 Acq Knowledge software를이용하였다. 량을합하여계산하였다. 배뇨간격 (micturition interval: MI) 은세번의배뇨주기중에서배뇨압과바로그다음배뇨주기의배뇨압사이의시간간격을측정하고그시간간격에서잔뇨를측정한시간을뺀값으로정의하였다. 배뇨주기들을 3 6분동안측정하였고, 측정된배뇨주기에서가장대표되는 3개의연속된주기를선택하여각변수의평균값들을측정하였다. 6) 자료분석및통계결과는평균값 ± 표준편차로표시하였으며, 정규분포는 Shapiro-Wilks W test를이용하였다. Student s t-test 와 Bonferroni correction을이용하여분석하였고, p값이.5,.1 미만인경우로나누어통계적으로유의한것으로판단하였다. 결과 1) Tolterodine 경구투여군 SHR에서 tolterodine 1 mg/kg을경구투여한결과 BP, TP, MP 등압력변수들및 BC, VV, RV 등의용적변수들에서통계적으로유의한차이를보이지않았다. 배뇨간격인 MI는 3.72±.78 ml에서 4.31±.72 ml 로증가되는경향은보였지만통계적인차이를보이지는않았다 (Table 1, Fig. 1). 5) 요역동학검사의측정변수들 2) Tolterodine 정맥주사군 세번의반복적인배뇨주기에서방광내압지표인기저압 (basal pressure: BP) 은방광내압검사에서가장낮은압력으로, 임계압 (threshold pressure: TP) 은방광수축직전의압력으로, 배뇨압 (micturition pressure: MP) 은배뇨시최대방광내압으로각각정의하였다. 용적변수지표인배뇨량 (voided volume: VV) 은요채집컵을 force displacement transducer에연결하여모인소변양으로측정하였고, 잔뇨량 (residual volume: RV) 은배뇨가끝난후방광에삽입된관을일정시간동안쥐보다낮은높이로유치시켜흘러나온식염수양으로측정하였으며, 방광용적 (bladder capacity: BC) 은잔뇨량과배뇨 SHR에서 tolterodine.3 mg/kg을정맥주사한결과 BP, MP의압력변수는통계적으로의미있게감소하였으나 TP는통계적으로유의한차이를보이지않았다. 용적변수인 RV는변화가없었으며 BC는.9±.13 ml 에서.95±.12 ml로, MI는 5.54±.7 ml에서 6.39±.72 ml로증가하는양상을보였지만통계적인차이를보이지않았다. (Table 2, Fig. 2). 고찰쥐를이용한과민성방광의실험동물모델로는방 154 대한배뇨장애요실금학회지

4 비마취하자연발생고혈압쥐에서 tolterodine 의경구및주사효과 Table 1. Pressure-Volume parameters during cystometry in awake, freely moving, spontaneous hypertensive rats (SHRs), which was administrated tolterodine orally. IVP BP. cmh 2O TP. cmh 2O MP. cmh 2O BC. ml VV. ml RV. ml MI. min SHR control (N=5) 19.9± ± ±3.3.61±.16.61± ±.78 SHR oral tolterodine (N=5) 18.9± ± ±4.9.68±.12.68± ±.72 IVP: Intra-vesical pressure, BP: Basal Pressure, TP: Threshold Pressure, MP: Micturition Pressure, BC: bladder capacity, VV: voided volume, RV: residual volume, MI: micturition interval. Results are expressed as mean±standard error of the mean. *p<.5, **p<.1 (unpaired Student s t test), versus toleterodine-administrated SHR. Tolterodine oral 1 mg kg -1 b.w. 6 IVP cm H 2O 4 Vol ml 2 min Fig. 1. The representative tracings of awake cystometry in Spontaneous hypertensive rat (SHR) before and after the oral administration of tolterodine 1 mg/kg. The cystometry after oral administration was performed one hour later, which was determined by the pharmacokinetic-pharmacodynamic (PK-PD) profile of toleterodine. There was no significant changes in pressure and volume parameters of cystometry between before and after the medication. 광의내재성물질로알려진 PGE 2 를이용하여과민성방광을유발시키는모델 [1,11], 방광출구폐색을통해유발되는과민성방광모델 [12,13], 유전적으로개발된고혈압쥐에서발생하는과민성방광모델 [8] 등세가지가가장많이사용되고있지만, 아직어떤모델이사람의과민성방광을가장잘반영하는지는확실히밝혀진바없다. 그러나최근에는 SHR이많은이들에게공식적으로대표적과민성방광모델로인정받고있는추세이다 [14]. 일반적으로방광수축은무스카린수용체를매개로 하여일어나는데, 과민성방광의핵심병태생리학적기전인저장기배뇨근과활동성또한이수용체를통하여발생하게된다. 무스카린수용체의세부형태 5가지중 M2와 M3 수용체가방광에중요한역할을하는데, M3 수용체는정상방광에서직접적인수축을일으키며 [15,16], 이수축은 rho kinase의활성과함께 L-type 칼슘채널을통한외부칼슘의유입에의존하는것으로알려져있다 [17]. M2 수용체는 M3 수용체와달리정상상태에서는직접적인역할을하지않고, 병리학적상태에서중요한직접적수축을일으키는 Vol. 13, No. 2,

5 이소영 진롱후 권용현 장진혁 강용진 윤상민 이택 Table 2. Pressure-Volume parameters during cystometry in awake, freely moving, spontaneous hypertensive rats (SHRs), which was administrated tolterodine intravenously. IVP BP. cmh 2O TP. cmh 2O MP. cmh 2O BC. ml VV. ml RV. ml MI. min SHR control (N=5) 1.±1.1* 3.9± ±3.**.9±.13.9± ±.7 SHR oral tolterodine (N=5) 8.7± ±2.6 41±6.4.95±.12.95± ±.72 IVP: Intra-vesical pressure, BP: Basal Pressure, TP: Threshold Pressure, MP: Micturition Pressure, BC: bladder capacity, VV: voided volume, RV: residual volume, MI: micturition interval. Results are expressed as mean±standard error of the mean. *p<.5, **p<.1 (unpaired Student s t test), versus toleterodine-administrated SHR. 6 IVP Tolterodine intravenous.3 mg kg -1 b.w. cm H 2O 4 Vol ml 2 min Fig. 2. The representative tracings of awake cystometry in Spontaneous hypertensive rat (SHR) before and after the intravenous administration of tolterodine.3 mg/kg. The basal pressure and micturition pressure was decreased after the medication, and in this rat, there showed significant changes in volume parameters of bladder capacity and micturition interval. However, in all data of five rats, there was no statistically significant change in volume parameters between before and after the medication. 것으로알려져있다 [18]. 또한이반응은 camp와관련된기전을통하여 beta-adrenoceptor의이완작용을억제하여간접적인수축반응을나타나는것으로밝혀져있다 [19]. 비마취하 SHR에서항콜린성제제인 tolterodine을경구와정맥주사를통해투여한뒤시행한요역동학검사결과방광의용량이나배뇨간격을늘리지는못하였으나, 정맥주사로투여한경우방광의기저압과배뇨압이감소되었는데, 이는기존의연구결과와유사한것이다 [4,6,7,9]. 항콜린성제제는방광에서 M3 수용체를억제하여방광수축을억제하기도하지만, 뇌혈관장벽 (brainblood barrier: BBB) 을통과하여중추신경계에있는무스카린수용체를통하여신경계에영향을나타낼가 능성이있다 [2]. 항콜린성제제가중추신경계에미치는영향에대한여러연구들이보고되어왔다 [2,21]. Ishiura 등 [22] 은무스카린수용체 agonist인 oxotremorine methiodide를비마취하정상쥐에정맥이나척수강내로주입하였을때는, 방광용적이투여량에따라증가하였으나, 무스카린수용체억제제인 atropine 을척수강내로주입하였을때는방광용적은변화가없고, 정맥을통해고용량으로주입하였을때만방광용적이증가한다고하였다. Abrams 등 [2] 은 oxybutynin이나 tolterodine을정맥주사하였을때방광용적에는영향이없었다고하였고, 반대로뇌일부에문제가있는비마취하쥐에서 oxybutynin을경구투여하였을때방광용적이증가되었다고보고하였다. 이렇듯항콜린성제제를비마취하에서사용하였을때방광의 156 대한배뇨장애요실금학회지

6 비마취하자연발생고혈압쥐에서 tolterodine 의경구및주사효과 무스카린수용체뿐만아니라중추신경계의무스카린수용체에작용하며또한중간의복잡한과정의변화에의해영향을받으므로, 배뇨압에는영향을미칠수있지만, 과민성방광의치료제로서가장중요하게여겨지는배뇨간격의변화는나타나지않을수있다는것을명심하여야한다. 과민성방광은다른질환과는달리환자의주관적증상을기초로하는질환이다. 이는과민성방광의가장근본적인기전인방광의불수의적수축즉배뇨근과활동성이중추신경계의영향에따라증상으로나타나거나혹은나타나지않을수도있다는의미이다. 동물을이용한연구에서비마취하상태가중요하긴하지만, 약물이방광뿐만아니라중추신경계에도영향을미치고, 이런영향에의해다른결과가도출될수있다는것을주지하여야한다. 오랜임상경험에따르면과민성방광환자에게항콜린성제제를투여하였을때, 배뇨간격의변화는약제투여후바로나타나는것이아니라, 일정기간이지난뒤나타나는것을관찰할수있다 [7,9]. 이는배뇨의변화를인지하고증상으로나타나게하는뇌의활동방식에따라배뇨증상의변화가바로나타날수도있지만 (Fig. 2), 증상의변화가방광뿐아니라방광을지배하는중추및말초신경계의만성적변화를통해서일어날가능성을의미하는것이다. 따라서이러한경우에는본연구의결과와같이즉각적인호전을보이지않을가능성이높다. 그러므로동물을이용한실험에서와같이약물투여후즉각적으로결과를관찰하는방식에서는아무리효과가좋은항콜린성제제라하더라도즉각적인배뇨간격의호전을보이기어려우므로, 향후장기적관점에서의약물의효과를관찰할수있는모델개발도병행되어야할것으로사료된다. 결론과민성방광모델인 SHR에서마취를하지않고 tolterodine 고용량을경구및정맥주사로투여한후관찰한결과정맥주사로투여한경우과민성방광에대한일부변수에서치료효과를보였지만, 경구로투여한경우모든변수에서치료효과를보이지않았다. 이는과민성방광의치료를위해개발된새로운약물의효 과를관찰하기위해, SHR을이용하여마취를하지않고경구로약물을투여하는것이정확한결과를도출해낼수없을가능성을시사한다. 그러므로과민성방광에대한항콜린성제제의약물효과를정확하게판단하기위하여는다른동물모델을추가적으로확립할필요가있을것으로사료된다. References 1) Hu TW, Wagner TH. Economic considerations in overactive bladder. Am J Manag Care 2;6: ) Grocela JA, Kanji A, Ternullo J. Prediction of Medicare drug formulary drugs for treatment of overactive bladder. J Urol 26;176: ) Hashim H, Abrams P. Drug treatment of overactive bladder: efficacy, cost and quality-of-life considerations. Drugs 24;64: ) Scarpero HM, Dmochowski RR. Muscarinic receptors: what we know. Curr Urol Rep 23;4: ) Alberts P. Classification of the presynaptic muscarinic receptor subtype that regulates 3H-acetylcholine secretion in the guinea pig urinary bladder in vitro. J Pharmacol Exp Ther 1995;274: ) Angelico P, Velasco C, Guarneri L, Sironi G, Leonardi A, Testa R. Urodynamic effects of oxybutynin and tolterodine in conscious and anesthetized rats under different cystometrographic conditions. BMC Pharmacol 25;5:14 7) Andersson KE. Overactive bladder--pharmacological aspects. Scand J Urol Nephrol Suppl 22;21: ) Jin LH, Andersson KE, Kwon YH, Park CS, Yoon SM, Lee T. Substantial detrusor overactivity in conscious spontaneously hypertensive rats with hyperactive behaviour. Scand J Urol Nephrol 29;43:3-7 9) Andersson KE. Antimuscarinics for treatment of overactive bladder. Lancet Neurol 24;3: ) Takeda H, Yamazaki Y, Igawa Y, Kaidoh K, Akahane S, Miyata H, et al. Effects of beta(3)-adrenoceptor stimulation on prostaglandin E(2)-induced bladder hyperactivity and on the cardiovascular system in conscious rats. Neurourol Urodyn 22;21: ) Meini S, Lecci A, Cucchi P, Catalioto RM, Criscuoli M, Maggi CA. Inflammation modifies the role of cy- Vol. 13, No. 2,

7 이소영 진롱후 권용현 장진혁 강용진 윤상민 이택 clooxygenases in the contractile responses of the rat detrusor smooth muscle to kinin agonists. J Pharmacol Exp Ther 1998;287: ) Berggren T, Uvelius B. Cystometrical evaluation of acute and chronic overdistension in the rat urinary bladder. Urol Res 1998;26: ) Mostwin JL, Karim OM, van Koeveringe G, Brooks EL. The guinea pig as a model of gradual urethral obstruction. J Urol 1991;145: ) Steers WD, Clemow DB, Persson K, Sherer T, Andersson KE, Tuttl JB. Observations from the spontaneously hypertensive rat. Insight into NGF regulation and noradrenergic hyper-innervation in the lower urinary tract. Adv Exp Med Biol 1999;462: ) Wang P, Luthin GR, Ruggieri MR. Muscarinic acetylcholine receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins. J Pharmacol Exp Ther 1995;273: ) Fetscher C, Fleichman M, Schmidt M, Krege S, Michel MC. M(3) muscarinic receptors mediate contraction of human urinary bladder. Br J Pharmacol 22;136: ) Schneider T, Fetscher C, Krege S, Michel MC. Signal transduction underlying carbachol-induced contraction of human urinary bladder. J Pharmacol Exp Ther 24;39: ) Pontari MA, Braverman AS, Ruggieri MR, Sr. The M2 muscarinic receptor mediates in vitro bladder contractions from patients with neurogenic bladder dysfunction. Am J Physiol Regul Integr Comp Physiol 24;286: ) Yamanishi T, Chapple CR, Yasuda K, Chess-Williams R. The role of M(2)-muscarinic receptors in mediating contraction of the pig urinary bladder in vitro. Br J Pharmacol 2;131: ) Abrams P, Andersson KE, Buccafusco JJ, Chapple C, de Groat WC, Fryer AD, et al. Muscarinic receptors: their distribution and function in body systems, and the implications for treating overactive bladder. Br J Pharmacol 26;148: ) Ishizuka O, Gu BJ, Yang ZX, Nishizawa O, Andersson KE. Functional role of central muscarinic receptors for micturition in normal conscious rats. J Urol 22;168: ) Ishiura Y, Yoshiyama M, Yokoyama O, Namiki M, de Groat WC. Central muscarinic mechanisms regulating voiding in rats. J Pharmacol Exp Ther 21; 297: 대한배뇨장애요실금학회지

페링야간뇨소책자-내지-16

페링야간뇨소책자-내지-16 야간뇨의진단과치료 - 실제적접근 - Reference 1. Choo MS, Ku JH, Park CH et al. Prevalence of Nocturia in a Korean Population Aged 40 to 89 Years. Neurourol Urodyn 2008; 27:60-64. 2. Weiss JP. Prevalence