兽研简要概述
本研究探讨了全身麻醉方式的选择是否会影响猫在卵巢子宫切除术中对托芬那酸（一种非甾体抗炎药）的体内处置过程。我们比较了两种标准麻醉方案：赛拉嗪-氯胺酮和丙泊酚-异氟烷。虽然两组药物吸收入血的总量（总体全身暴露量）相似，但吸收速率存在显著差异。接受丙泊酚-异氟烷麻醉的猫皮下药物吸收速度比接受赛拉嗪-氯胺酮麻醉的猫更快。主要临床意义在于，兽医可以安全地使用任一种麻醉方案给予标准皮下剂量（4 mg/kg）的托芬那酸。由于总体全身暴露量保持稳定，无需调整剂量，从而确保猫手术患者术后疼痛管理有效且简便。
1. 引言
卵巢子宫切除术（即绝育术）是小动物临床中常见的外科手术，涉及切除子宫和卵巢[1]。该手术后，触发动物发情的激素水平将下降，规律的发情周期将不再继续。卵巢子宫切除术消除了发情行为、出血和意外妊娠的可能性[2]。它也是卵巢和子宫肿瘤及子宫蓄脓等疾病的治疗选择[3]。卵巢子宫切除术会引起疼痛和炎症，有效治疗对于减轻疼痛、加速康复和减少不良并发症至关重要[4]。
非甾体抗炎药（NSAIDs）具有镇痛、抗炎和解热作用，在临床中具有优势。这些药物的药理作用源于通过环氧化酶（COX）酶抑制花生四烯酸生成前列腺素[5]。COX有两种类型：COX-1和COX-2。COX-1在大多数组织中持续表达，在调节肾血流、保护胃肠道黏膜和维持稳态等生理过程中发挥作用。虽然COX-2在肾脏和生殖系统中持续表达，但它在炎症区域特别可被诱导，并在炎症过程中发挥作用[5,6]。NSAIDs对COX的作用在不良反应方面意义重大，对胃肠道和肾脏系统及稳态的不良反应被认为主要源于COX-1抑制[4,5]。NSAIDs常用于减轻卵巢子宫切除术引起的疼痛和炎症[1,7]。然而，由于对毒性的担忧、成本限制和数据不足，获准用于猫的NSAIDs数量有限[5]。
托芬那酸是芬那酸类NSAID，具有镇痛、解热和抗炎特性，以及抗内毒素、抗癌、促进阿尔茨海默病和抗菌活性[8,9]。对COX酶的作用因动物物种而异；在山羊中作为选择性COX-2抑制剂，但在犊牛中表现出非选择性抑制作用[10,11]。在犬中表现出对COX-2酶的选择性作用[12,13]。托芬那酸在土耳其、欧洲、新西兰、加拿大和澳大利亚获准用于猫[5]。在猫中用于发热、术后疼痛、急性和慢性炎症性疾病及下呼吸道疾病[5,14]。据报道，它能成功减轻猫卵巢子宫切除术引起的疼痛，其效果与美洛昔康、卡洛芬、酮洛芬和罗贝那考昔等其他NSAIDs相似[4,15,16]。
全身麻醉的主要目标是使患者意识消失、抑制疼痛感知并调节自主反射[17]。猫卵巢子宫切除术最常用的全身麻醉方案是赛拉嗪-氯胺酮和丙泊酚-异氟烷。氯胺酮是一种分离性全身麻醉药，可提供遗忘、镇痛和制动。然而，由于它不能提供足够的肌肉松弛，常与赛拉嗪联合使用，后者是一种α2肾上腺素受体激动剂，具有肌肉松弛、镇痛、镇静和抗焦虑等作用[18,19]。异氟烷是一种常见的吸入麻醉药，其特征是代谢率极低和血气溶解度低，而丙泊酚是一种短效静脉麻醉药，常用于全身麻醉的诱导和维持。这两种药物常联合使用以增强麻醉效果并减少副作用[20]。
尽管药品说明书附页中关于托芬那酸在猫体内药代动力学信息有限[14]，但据我们所知，尚未开展详细的药代动力学研究。托芬那酸的药代动力学已在多种动物物种中进行了考察，显示出显著的种间差异[6,9,21,22,23,24]。药代动力学有助于定量评估药物吸收、分布、代谢和排泄过程[25]。全面了解目标物种的这些药代动力学参数对于确定临床实践中正确的给药策略至关重要[26]。此外，对于推荐使用该药物的疾病或状况，开展药物药代动力学研究对于有效治疗至关重要[26]。尽管托芬那酸在猫卵巢子宫切除术中得到了有效应用，但缺乏其药代动力学数据。据报道，麻醉药物可改变手术过程中同时使用的其他药物的药代动力学[27]。麻醉药物对生理参数的影响各不相同[28]，因此，药物药代动力学的变化可能因麻醉方案而异。本研究假设，与丙泊酚-异氟烷麻醉相比，赛拉嗪-氯胺酮麻醉的独特血流动力学和生理效应会引起接受卵巢子宫切除术的猫皮下注射托芬那酸的吸收和处置动力学发生显著改变。本研究的目的是测定在赛拉嗪-氯胺酮和丙泊酚-异氟烷麻醉下进行卵巢子宫切除术期间，猫皮下注射4 mg/kg剂量托芬那酸的药代动力学。
2. 材料与方法
2.1. 化学品
纯度至少为98%的托芬那酸分析标准品购自Sigma Aldrich（美国密苏里州圣路易斯）。用于该药物分析的化学品均为HPLC级。乙腈和甲醇由Chemlab（比利时泽德尔海姆）提供，正磷酸由Sigma Aldrich（美国密苏里州圣路易斯）提供。使用托芬那酸的注射用制剂（Tolfenak Injection Solution，40 mg/mL，Alke，土耳其伊斯坦布尔）对猫给药。使用赛拉嗪（Xylazin Bio 2%；Bioveta，捷克共和国伊万诺维采-纳哈内）-氯胺酮（Ketasol 10%，VetViva Richter GmbH，奥地利韦尔斯）和丙泊酚（Propofol-Pf Mct 1% Injection/Infusion Emulsion；Polifarma，土耳其泰基尔达）-异氟烷（Vetflurane，Virbac，法国卡罗）麻醉商品的制剂进行麻醉诱导。
2.2. 动物
本研究使用12只健康的雌性家猫（2-4岁；体重2.0-3.4 kg）。这些猫选自被送至锡尔特大学动物健康应用与研究医院产科和妇科进行择期卵巢子宫切除术的猫。纳入研究的猫为发情后6-10周、体格检查临床健康、血液学分析、血清生化（肝肾功能检查）、阴道细胞学和腹部超声检查结果正常的猫。研究排除标准包括有慢性疾病史、肝或肾功能衰竭、血液学异常、妊娠、哺乳、麻醉禁忌症以及最近30天内使用过NSAIDs或皮质类固醇的猫。猫主人被告知麻醉程序和研究内容，并获得了书面知情同意。猫在手术前一天住院，术前禁食12小时。本研究经锡尔特大学当地伦理委员会批准（方案编号：2024/07-47）。
2.3. 实验设计
使用计算机生成的随机化方法将猫随机分配到两个相等组（每组n = 6）：第1组接受赛拉嗪-氯胺酮麻醉，第2组接受丙泊酚-异氟烷麻醉。由一名外科医生在一个月内通过标准腹中线入路完成所有卵巢子宫切除术。
第1组（X/K）：首先，以4 mg/kg剂量对猫皮下注射托芬那酸。麻醉诱导时，在托芬那酸给药十分钟后以1 mg/kg剂量肌肉注射赛拉嗪，十分钟后以10 mg/kg剂量注射氯胺酮。氯胺酮给药十分钟后开始手术操作。因此，托芬那酸在手术操作前30分钟给药。
第2组（P/I）：首先，以4 mg/kg剂量对猫皮下注射托芬那酸。麻醉诱导时，在托芬那酸给药十五分钟后以6 mg/kg剂量静脉注射丙泊酚。麻醉维持时，在丙泊酚给药10分钟后进行气管插管，通过半封闭重呼吸系统给予2-3%异氟烷 in 氧气。异氟烷麻醉约5分钟后开始手术操作。因此，托芬那酸在手术操作前30分钟给药。
2.4. 手术操作
进行了眼睑反射和踏板反射检查，当猫完全松弛且下颌张力消失时，认为诱导完成。在确认符合正常无菌手术规程后，对所有猫通过腹中线剖腹术进行卵巢子宫切除术。使用可吸收缝线材料（polyglactin 910）以改良Miller结技术结扎卵巢蒂和子宫体。记录从皮肤切开到皮肤闭合的手术持续时间，为40–45分钟。手术过程中，每只猫接受约50–75 mL等渗溶液进行液体支持。术后疼痛和恢复由主治兽医（O.Y.K.和A.U.）作为常规护理的一部分进行临床评估。必要时可获取补救镇痛；然而，没有猫需要补救镇痛。
2.5. 给药与采血
两组均在手术开始前30分钟皮下（肩胛之间）给予托芬那酸，剂量为4 mg/kg。为采集血样，在手术前一天于镇静状态下将22号导管无菌置入前臂静脉。在给药后0（对照，托芬那酸给药前）、15、30和45分钟，以及1、2、4、6、8、10、12、24和48小时将血样采集至锂肝素管中。根据每只猫调整采血量，确保在研究期间采集的血液不超过总血量的10%（每次采样最多0.6–1 mL）。每次采集后，用0.6–1 mL肝素化生理盐水冲洗导管。肝素管通过倒置混匀，然后在1小时内离心（4000× g离心10分钟）。离心后，将血浆样品转移至微量离心管中，然后储存于−80 °C直至分析。
2.6. 托芬那酸分析
采用高效液相色谱法（HPLC）结合紫外（UV）检测测定血浆托芬那酸浓度，使用先前验证的方法并稍作修改[21]。简言之，为使蛋白质变性，向100 µL血浆样品中加入150 µL乙腈。混合物涡旋30秒，并以12,000× g离心10分钟。将上清液转移至自动进样瓶，取等分试样（20 µL）注入HPLC系统（Shimadzu，京都，日本）。使用InertSustain C18柱（250 × 4.6 mm内径，5 μm粒径，GL Sciences，东京，日本）在40 °C和289 nm波长下进行托芬那酸的色谱分离。流动相由乙腈和0.1%正磷酸（65:35 v/v%）组成，流速为1 mL/min。数据使用LC Solution软件程序（版本1.25 SP5）处理。
2.7. 分析方法验证
根据欧洲药品管理局指南[29]，在血浆中验证了HPLC方法的线性、检测限（LOD）和定量限（LOQ）、回收率以及日内和日间准确度。线性通过使用对照血浆样品构建的校准曲线进行线性回归分析评估，所述样品加标托芬那酸浓度为0.04至20 μg/mL。LOD定义为产生信噪比为3的托芬那酸血浆浓度，LOQ确定为产生信噪比为6的托芬那酸血浆浓度。在五个不同日期对加标三种不同浓度（0.1、1和10 μg/mL）托芬那酸的血浆各分析三次，以评估回收率和日内及日间精密度。然后将这些浓度与工作标准品比较以确定回收率。计算精密度并以变异系数（CV%）表示。
2.8. 药代动力学分析
通过WinNonlin 6.1.0.173软件（Pharsight；Certara，圣路易斯，密苏里州，美国）采用非房室分析法从每只猫的血浆托芬那酸浓度获得药代动力学参数。末端消除半衰期（t1/2λz）通过公式t1/2λz = ln(2)/λz确定。曲线下面积（AUC）通过线性向上/对数向下方法测量。计算平均驻留时间（MRT）、表观分布容积（Vdarea/F）和表观总清除率（ClT/F）。确定从最后可测浓度外推至无穷大的AUC占总AUC的百分比（AUCextrap%）。峰血浆浓度（Cmax）和达峰时间（Tmax）直接由观测数据确定。
2.9. 统计分析
使用SPSS 22.0软件（IBM Corp.，阿蒙克，纽约，美国）进行统计分析。p值<0.05被认为具有统计学显著性。药代动力学参数以几何均值（最小值-最大值）报告，而Tmax以中位数（最小值-最大值）报告。使用Shapiro–Wilk检验验证药代动力学参数的正态分布。进行独立t检验以分析治疗组之间的统计学显著差异。
3. 结果
3.1. 分析方法验证
该方法具有良好的特异性，在托芬那酸的保留时间处色谱图中无任何峰出现。工作曲线和校准曲线在0.04–20 μg/mL浓度范围内线性关系良好，R2值均>0.999。LOD为0.02 μg/mL，LOQ为0.04 μg/mL。提取过程高效，托芬那酸的平均回收率高于95%。日内和日间变异系数分别<6.88%和<5.49%。日内和日间偏差分别为±4.15%和±3.23%。总体而言，该验证方法已被证明可可靠地用于采用HPLC测定猫血浆中的托芬那酸。
3.2. 药代动力学
图1展示了在不同麻醉方案下接受卵巢子宫切除术的猫皮下注射4 mg/kg剂量托芬那酸后的半对数血浆浓度-时间曲线。在两个麻醉组中，托芬那酸均可检测至24 h。表1列出了非房室药代动力学参数。接受赛拉嗪-氯胺酮麻醉的卵巢子宫切除术猫中，托芬那酸的t1/2λz和MRT0-∞长于接受丙泊酚-异氟烷麻醉的猫。与接受赛拉嗪-氯胺酮麻醉的猫相比，接受丙泊酚-异氟烷麻醉的猫表现出更大的Cmax和更短的Tmax（p < 0.05）。两个麻醉组的AUC、Vdarea/F和ClT/F相当（p > 0.05）。
4. 讨论
本研究首次揭示了在赛拉嗪-氯胺酮和丙泊酚-异氟烷麻醉下接受卵巢子宫切除术的猫中托芬那酸的药代动力学。研究得出结论，托芬那酸的药代动力学参数（如t1/2λz、Cmax和Tmax）因麻醉方案不同而变化。
总体而言，葡萄糖醛酸化代谢在NSAIDs的消除中起着关键作用。由于猫肝脏葡萄糖醛酸化能力较低，这些药物在猫体内的分布与犬不同[5]。已有报道称，NSAIDs的t1/2λz（尤其是那些通过葡萄糖醛酸化反应代谢的药物）在猫体内长于犬[5]。因此，NSAIDs在猫体内具有显著的毒性潜力，以适当的剂量和给药间隔使用此类药物对安全性至关重要[30,31]。NSAIDs的不良反应尤其与胃肠道、肾脏和肝脏系统相关[5]。托芬那酸的胃肠道损伤风险低于许多其他NSAIDs，且在治疗剂量下耐受性良好[32,33,34]。同样，以4 mg/kg剂量口服托芬那酸14天对猫的肾功能无不良影响[35]。以4 mg/kg剂量给予托芬那酸3天未引起猫腹泻、呕吐、多尿、多饮或攻击行为的任何变化[5]。
托芬那酸在猫中的推荐剂量为4 mg/kg，经口服或皮下途径给药[5,14]。对接受卵巢子宫切除术的猫以4 mg/kg剂量皮下给予托芬那酸并显示出有效性[15,16]。对于猫的卵巢子宫切除术，通常建议单次注射一剂NSAID[7]。推荐术前镇痛治疗，因为它可以导致更高的血浆和组织液/炎性渗出液水平，或通过减少外周产生的有害信息量来减轻中枢变化[36,37]。因此，在本研究中，于卵巢子宫切除术前30 min皮下给予单剂4 mg/kg托芬那酸。
手术操作和麻醉药物会引起机体生理改变。手术引起与疼痛、焦虑、心血管不稳定和血流动力学变化相关的应激，触发儿茶酚胺、皮质类固醇和细胞因子的释放，进而启动一系列可持续数天的复杂代谢事件[38]。麻醉药物改变机体灌注、肝脏和代谢功能、肾功能以及药物与血浆蛋白的结合[27]。因此，手术操作可能导致同时使用药物的药代动力学差异[39]。卵巢子宫切除术改变了美洛昔康的处置，并延迟了卡洛芬在犬体内的消除[36,40]。
t1/2λz定义为药物在体内的浓度降至初始量一半所需的时间[41]。接受卵巢子宫切除术的猫中，托芬那酸的t1/2λz在赛拉嗪-氯胺酮组中长于丙泊酚-异氟烷麻醉组。然而，两个麻醉组之间的差异仅为11%，可能无临床意义。t1/2λz是由清除率和分布容积共同衍生的参数，受这些参数变化的影响[42]。本研究中两个麻醉组的Vdarea/F和ClT/F值相当。由于吸收过程不会产生影响，静脉给药是测定这两个参数最合适的途径。在本研究中，由于托芬那酸仅通过皮下途径给药，无法确定不同麻醉组之间吸收的差异。尽管两个麻醉组中这两个参数相似，但t1/2λz的变化可能是由于麻醉组之间吸收的差异所致。
在右美托咪定-氯胺酮和丙泊酚-异氟烷麻醉下接受卵巢子宫切除术的猫中，托芬那酸的Vdarea/F和ClT/F相似。NSAIDs由于与血浆蛋白显著结合且在血流中呈离子化状态，通常具有有限的分布容积。芬那酸衍生物如托芬那酸的pKa值约为4，在生理pH水平（血液=7.4）下大部分呈离子化[43]。托芬那酸在多种动物物种（包括犬）中与血浆蛋白高度结合[21]。因此，托芬那酸通常具有较低的分布容积[44]。托芬那酸经肝脏代谢，并随其代谢物通过尿液和胆汁排泄[45,46]。据报道，其在猫体内主要以原形排泄[14]。据报道，外科手术和麻醉药会影响心输出量、组织灌注、肾和肝血流量、生物转化酶活性以及血浆蛋白水平，这可能影响药物的分布容积和清除率[27,39,47]。由于本研究中没有未进行手术的对照组，因此无法评估麻醉方案对此的影响。然而，在接受卵巢子宫切除术的犬中，静脉注射后美洛昔康的Vd值降低，而Cl值保持不变[40]。右美托咪定-氯胺酮和异氟烷麻醉对血流动力学和心脏功能的影响[28]、右美托咪定-氯胺酮和氯胺酮-丙泊酚麻醉对脉率、直肠温度和血液生化指标的影响[48]，以及美托咪定、右美托咪定-氯胺酮-氟烷和丙泊酚对肾小球滤过率的影响各不相同[49]。这些结果表明，病理生理效应因麻醉方案而异。据我们所知，尚无研究比较右美托咪定-氯胺酮和丙泊酚-异氟烷麻醉方案的病理生理效应；然而，由于这两种麻醉方案表现出相似的效果，这些组间的Vdarea/F和ClT/F可能相似。
与右美托咪定-氯胺酮麻醉的动物相比，丙泊酚-异氟烷麻醉的猫中托芬那酸的Cmax更高，Tmax更短。这表明在丙泊酚-异氟烷麻醉后，托芬那酸在更短时间内达到更高的血药浓度。Cmax反映药物从注射部位吸收的速率和程度与消除速率之间的关系[50]。药物的吸收速率和程度可因药物制剂、注射部位和组织灌注而异。两组使用相同制剂的托芬那酸，且注射部位相同。因此，这些参数的变化可能尤其源于组织灌注的变化。在外科手术过程中，心输出量减少和外周灌注降低会减少药物从注射部位的吸收，这可能导致Cmax降低和Tmax延长[27]。麻醉药对血流动力学和心脏功能的影响各不相同[28]。与异氟烷麻醉相比，右美托咪定-氯胺酮麻醉引起血流动力学、心脏功能以及糖脂代谢的更多变化[28]。右美托咪定-氯胺酮麻醉后15分钟心率显著下降，但在40分钟时与异氟烷相似[28]。在本研究中，托芬那酸Cmax和Tmax值的差异可能是由于各麻醉组之间血流动力学和心脏功能的差异。然而，应注意此评估仅为解释，因为本研究未评估麻醉药对血流动力学和心脏功能的影响。
在右美托咪定-氯胺酮和丙泊酚-异氟烷麻醉下接受卵巢子宫切除术的猫中，托芬那酸的AUC相似。在两个麻醉组中托芬那酸均可检测至24小时。AUC代表药物随时间推移的总全身暴露量，全身清除率和生物利用度在决定AUC中起关键作用（剂量×F/Cl）[51,52]。两个麻醉组中AUC的相似性可能是由于全身清除率与生物利用度之间的关系。两个麻醉组中AUC值相似可能表明托芬那酸的全身暴露量相似，且可与两种不同的麻醉方案配合使用。
据我们所知，关于托芬那酸在猫体内剂量-浓度关系的药效学研究不足；因此，无法比较血浆浓度与药效学疗效之间的关系。此外，据报道NSAIDs的血浆浓度并不反映其有效性[53]。血浆蛋白倾向于在炎症区域积聚，而与此类蛋白高度结合的药物（如托芬那酸）可在该区域积聚并发挥更持久的作用[10]。因此，需要建立接受卵巢子宫切除术的猫中托芬那酸的剂量-反应关系。
本研究的结果应在考虑某些局限性的前提下进行解读。首先，每组使用的动物数量相对较少（n = 6），可能限制了药代动力学结果的广泛推广。其次，由于没有非手术对照组，无法评估手术操作和麻醉方案相对于基线状态对托芬那酸药代动力学的绝对影响。第三，尽管吸收期的改变被归因于血流动力学变化，但手术期间未记录心输出量或血压等特定心血管参数。第四，由于包括托芬那酸在内的非甾体抗炎药倾向于在渗出液中蓄积，血浆浓度可能无法反映治疗效果。因此，需要开展表征托芬那酸在猫体内渗出液浓度和药代动力学的研究，以评估其治疗效果。第五，尽管对术后疼痛和恢复过程进行了临床评估，但未采用猫疼痛评分系统。因此，无法完全排除与疼痛相关的混杂效应，也无法评估疼痛严重程度与生物标志物之间的关系，这构成了一项局限性。此外，未在卵巢子宫切除术猫中测定托芬那酸的血浆蛋白结合率和代谢情况，这些数据本可提供更深入的机制性见解。最后，由于本研究仅聚焦于药代动力学，托芬那酸在这些麻醉方案下的临床疗效和剂量-反应关系仍有待未来研究评估。
5. 结论
本研究首次揭示了在不同麻醉方案下接受卵巢子宫切除术的猫皮下注射托芬那酸的药代动力学特征。尽管丙泊酚-异氟烷麻醉导致吸收期出现统计学显著变化——具体表现为更高的峰值Cmax和更短的达峰时间Tmax——但这些变化不太可能具有临床意义。两组之间总体全身暴露量（AUC）、表观清除率相当，且t1/2λz差异相对较小，表明无论选择何种麻醉方案，托芬那酸均可按标准4 mg/kg剂量安全有效地给药，无需调整剂量。然而，为充分优化猫科临床实践中的术后疼痛管理，未来研究应探讨托芬那酸在手术患者中的药效学疗效、剂量-反应关系及靶组织蓄积情况。此外，本研究未纳入仅接受任一麻醉方案而未进行手术操作的猫组。因此，难以区分所观察到的托芬那酸药代动力学变化是源于手术创伤/应激还是麻醉药物的影响。因此，需要开展纳入麻醉对照组的进一步研究。
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Simple Summary
This study investigated whether the choice of general anesthesia affects how the body processes tolfenamic acid, a nonsteroidal anti-inflammatory drug, in cats undergoing ovariohysterectomy. We compared two standard anesthetic protocols: xylazine–ketamine and propofol–isoflurane. While the total amount of the drug absorbed into the bloodstream (overall systemic exposure) was similar in both groups, the rate of absorption differed significantly. Cats that received propofol–isoflurane anesthesia absorbed the subcutaneous medication more rapidly than those receiving xylazine–ketamine. The main clinical implication is that veterinarians can safely administer the standard subcutaneous dose (4 mg/kg) of tolfenamic acid using either anesthetic protocol. Because the overall systemic exposure remains stable, no dosage adjustments are necessary, ensuring effective and simplified postoperative pain management for feline surgical patients.
1. Introduction
Ovariohysterectomy, or sterilization, is a commonly performed surgical operation in small animal practice that involves the removal of the uterus and ovaries [1]. Following this surgery, the levels of hormones that trigger heat in the animal will decrease, and the regular estrus cycle will not continue. Ovariohysterectomy eliminates the possibility of estrus behavior, bleeding, and unwanted pregnancy [2]. It is also a treatment option for conditions such as ovarian and uterine tumors and pyometra [3]. Ovariohysterectomy surgery causes pain and inflammation, and effective treatment is crucial in reducing pain, accelerating recovery, and minimizing unwanted complications [4].
Nonsteroidal anti-inflammatory drugs (NSAIDs) have analgesic, anti-inflammatory, and antipyretic effects, which provide an advantage in practice. The pharmacological effect of these drugs results from inhibiting the production of prostaglandins from arachidonic acid via cyclooxygenase (COX) enzymes [5]. There are two types of COX: COX-1 and COX-2. COX-1 is consistently expressed in most tissues and plays a role in physiological processes such as regulating renal blood flow, protecting the gastrointestinal mucosa, and maintaining homeostasis. Although COX-2 is constantly expressed in the kidney and reproductive system, it is particularly inducible in the inflammatory area and plays a role in inflammatory processes [5,6]. The effects of NSAIDs on COXs are significant in terms of adverse effects, and the adverse effects on the gastrointestinal and renal systems and homeostasis are thought to be primarily due to COX-1 inhibition [4,5]. NSAIDs are commonly used to reduce pain and inflammation caused by ovariohysterectomy [1,7]. However, the number of licensed NSAIDs for use in cats is limited due to concerns about toxicity, cost constraints, and insufficient data [5].
Tolfenamic acid is an NSAID from the fenamate category, exhibiting analgesic, antipyretic, and anti-inflammatory properties along with antiendotoxic, anticancer, Alzheimer’s disease-promoting, and antibacterial activities [8,9]. The effects on COX enzymes differ among animal species; it acts as a selective COX-2 inhibitor in goats, but it demonstrates a non-selective inhibitory effect in calves [10,11]. It exhibits a selective effect on COX-2 enzymes in canines [12,13]. Tolfenamic acid is approved for use in cats in Türkiye, Europe, New Zealand, Canada, and Australia [5]. It is used in cats for fever, postoperative pain, acute and chronic inflammatory conditions, and lower respiratory tract disorders [5,14]. It has been reported to successfully reduce pain caused by ovariohysterectomy surgery in cats, and its effect is similar to other NSAIDs such as meloxicam, carprofen, ketoprofen, and robenacoxib [4,15,16].
The main objective of general anesthesia is to induce unconsciousness in the patient, inhibit the perception of pain, and regulate autonomic reflexes [17]. The most frequent general anesthetic protocols used for ovariohysterectomy surgery in cats are xylazine–ketamine and propofol–isofulurane. Ketamine is a dissociative general anesthetic that provides amnesia, analgesia, and immobilization. However, because it does not provide sufficient muscle relaxation, it is used in combination with xylazine, an α2 adrenoreceptor agonist that has effects such as muscle relaxation, analgesia, sedation, and anxiolysis [18,19]. Isoflurane is a common inhalational anesthetic characterized by its minimal metabolic rate and low blood gas solubility, whereas propofol is a brief-acting intravenous anesthetic frequently employed for the initiation and sustenance of general anesthesia. These two drugs are often used in combination to enhance anesthetic efficacy and reduce side effects [20].
Although there is limited information in the drug prospectus inserts regarding the pharmacokinetics of tolfenamic acid in cats [14], to our knowledge, no detailed pharmacokinetic study has been conducted. The pharmacokinetics of tolfenamic acid have been examined across many animal species, demonstrating notable interspecies variations [6,9,21,22,23,24]. Pharmacokinetics facilitates the quantitative assessment of the processes involved in the absorption, distribution, metabolism, and excretion of medicines [25]. A comprehensive understanding of these pharmacokinetic parameters in the target species is crucial for determining correct dosage strategies in clinical practice [26]. Furthermore, in the diseases or conditions for which the drug use is recommended, conducting pharmacokinetic studies of the drugs is critical for effective treatment [26]. Despite the effective application of tolfenamic acid in cats’ ovariohysterectomy procedures, there is a lack of data regarding its pharmacokinetics. It has been reported that anesthetic drugs can alter the pharmacokinetics of other drugs used concurrently during surgical procedures [27]. The effects of anesthetic drugs on physiological parameters vary [28], and therefore, changes in the pharmacokinetics of the drug may differ depending on the anesthesia protocol. This study hypothesized that the distinct hemodynamic and physiological effects of xylazine–ketamine versus propofol–isoflurane anesthesia induce significant alterations in the absorption and disposition kinetics of subcutaneously administered tolfenamic acid in cats undergoing ovariohysterectomy. The aim of this study was to determine the pharmacokinetics of tolfenamic acid administered subcutaneously at a dose of 4 mg/kg during ovariohysterectomy performed under xylazine–ketamine and propofol–isoflurane anesthesia in cats.
2. Materials and Methods
2.1. Chemicals
A tolfenamic acid analytical standard with a purity of at least 98% was purchased from Sigma Aldrich (St. Louis, MO, USA). Chemicals used for the analysis of this drug were HPLC grade. Acetonitrile and methanol were supplied by Chemlab (Zedelgem, Belgium) and orthophosphoric acid by Sigma Aldrich (St. Louis, MO, USA). Parenteral (Tolfenak Injection Solution, 40 mg/mL, Alke, Istanbul, Türkiye) formulation of tolfenamic acid was used to administer the drug to cats. Anesthesia was induced with commercial preparations of xylazine (Xylazin Bio 2%; Bioveta, Ivanovice na Hané, Czech Republic)–ketamine (Ketasol 10%, VetViva Richter GmbH, Wels, Austria) and propofol (Propofol-Pf Mct 1% Injection/Infusion Emulsion; Polifarma, Tekirdağ, Türkiye)–isoflurane (Vetflurane, Virbac, Carros, France) anesthesia.
2.2. Animals
Twelve healthy female domestic cats (2–4 years old; body weight 2.0–3.4 kg) were used in this study. The cats were selected from those brought to the Obstetrics and Gynecology Department of Siirt University Animal Health Application and Research Hospital for scheduled ovariohysterectomy. Cats that were 6–10 weeks post-estrus, clinically healthy on physical examination, and had normal findings in hematological analysis, serum biochemistry (liver and kidney function tests), vaginal cytology, and abdominal ultrasonography were included in the study. Exclusion criteria for cats in the study included a history of chronic disease, liver or kidney failure, hematological abnormalities, pregnancy, lactation, contraindications to anesthesia, and use of NSAIDs or corticosteroids within the last 30 days. Cat owners were informed about the anesthesia procedures and the study, and written informed consent was obtained. Cats were hospitalized one day before surgery and fasted for 12 h prior to the procedure. The study was approved (protocol number: 2024/07-47) by the Local Ethics Committee of Siirt University.
2.3. Experimental Design
Cats were randomly allocated into two equal groups (n = 6 per group) using computer-generated randomisation: Group 1 received xylazine–ketamine anesthesia, and Group 2 received propofol–isoflurane anesthesia. A single surgeon performed all ovariohysterectomy procedures via a standard ventral midline approach within one month.
Group 1 (X/K): First, tolfenamic acid was administered to the cats subcutaneously at a dose of 4 mg/kg. For anesthesia induction, xylazine was administered intramuscularly at a dose of 1 mg/kg ten minutes after the tolfenamic acid administration, followed ten minutes later by ketamine at a dose of 10 mg/kg. The surgical procedure commenced ten minutes after the ketamine administration. Thus, tolfenamic acid was administered 30 min prior to the surgical procedure.
Group 2 (P/I): First, tolfenamic acid was administered to the cats subcutaneously at a dose of 4 mg/kg. For anesthesia induction, propofol was administered intravenously at a dose of 6 mg/kg fifteen minutes after tolfenamic acid administration. For anesthesia maintenance, 2–3% isoflurane in oxygen was administered via a semi-closed rebreathing system following endotracheal intubation 10 min after propofol administration. The surgical procedure began approximately 5 min after isoflurane anesthesia. Therefore, tolfenamic acid was administered 30 min before the surgical procedure.
2.4. Surgical Procedure
Palpebral and pedal reflex checks were performed, and induction was considered complete when the cat was fully relaxed and had lost lower mandibular tone. Following the confirmation of normal aseptic surgical protocols, ovariohysterectomy was performed on all cats via ventral midline laparotomy. The ovarian stalks and uterine body were ligated with absorbable suture material (polyglactin 910) using a modified Miller knot technique. The duration of the surgery, from skin incision to skin closure, was recorded as 40–45 min. During the procedures, each cat received approximately 50–75 mL of isotonic solution for fluid support. Postoperative pain and recovery were clinically assessed by the attending veterinarians (O.Y.K. and A.U.) as part of routine care. Rescue analgesia was accessible if necessary; yet, no cat needed rescue analgesia.
2.5. Drug Administration and Blood Sampling
Tolfenamic acid was administered subcutaneously (between the shoulder blades) at a dose of 4 mg/kg to both groups 30 min before the surgical procedure began. To collect blood samples, a 22-gauge catheter was aseptically placed in the antebrachial vein under sedation one day prior to surgery. Blood samples were collected into lithium heparin tubes at 0 (control, before tolfenamic acid administration), 15, 30 and 45 min, and 1, 2, 4, 6, 8, 10, 12, 24, and 48 h after drug administration. The volume of blood drawn was adjusted for each cat, ensuring that no more than 10% of the total blood volume was removed during the research duration (maximum of 0.6–1 mL each sample). Following each collection, the catheter was flushed with 0.6–1 mL of heparinised saline. The heparin tubes were mixed by inversion and then centrifuged (at 4000× g for 10 min) within 1 h. After centrifugation, plasma samples were transferred to microcentrifuge tubes and then stored at −80 °C until analysis.
2.6. Tolfenamic Acid Analysis
Plasma tolfenamic acid concentrations were determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection using a previously validated method with minor modifications [21]. Briefly, to denature the proteins, 150 µL of acetonitrile was added to 100 µL of plasma sample. The mixture was vortexed for 30 s and centrifuged at 12,000× g for 10 min. The supernatant was transferred to autosampler vials, and an aliquot (20 µL) was injected into the HPLC system (Shimadzu, Kyoto, Japan). Chromatographic separation of tolfenamic acid was performed using an InertSustain C18 column (250 × 4.6 mm inner diameter, 5 μm particle size, GL Sciences, Tokyo, Japan) maintained at 40 °C and a wavelength of 289 nm. The mobile phase consisted of acetonitrile and 0.1% orthophosphoric acid (65:35 v/v%), with a flow rate of 1 mL/min. The data were processed using the LC Solution software program (Version 1.25 SP5).
2.7. Validation of the Analytical Method
Linearity, limits of detection (LOD) and quantification (LOQ), recovery, and intra-day and inter-day accuracy of the HPLC method were verified in plasma according to the European Medicines Agency guidelines [29]. Linearity was assessed by linear regression analysis of calibration curves constructed using control plasma samples spiked with tolfenamic acid at concentrations from 0.04 to 20 μg/mL. The LOD was defined as the plasma concentration of tolfenamic acid that produces a signal-to-noise ratio of 3, and the LOQ was determined as the plasma concentration of tolfenamic acid that produces a signal-to-noise ratio of 6. Plasma spiked with tolfenamic acid at three different concentrations (0.1, 1 and 10 μg/mL) was analyzed three times on five separate days for assessing the recovery and intraday and interday precision. These concentrations were then compared to working standards to determine recovery. Precision was calculated and expressed as coefficient of variation (CV%).
2.8. Pharmacokinetic Analysis
Pharmacokinetic parameters were obtained from plasma concentrations of tolfenamic acid in each cat by a non-compartmental assay via WinNonlin 6.1.0.173 software (Pharsight; Certara, St. Louis, MO, USA). The terminal elimination half-life (t1/2λz) was determined by formulating t1/2λz = ln(2)/λz. Area under the curve (AUC) was measured by linear up/log down method. The mean residence time (MRT), apparent volume of distribution (Vdarea/F), and apparent total body clearance (ClT/F) were calculated. AUC extrapolated from the last measurable concentration to infinity as a percentage of total AUC (AUCextrap%) was determined. The peak plasma concentration (Cmax) and the time to reach Cmax (Tmax) were determined directly from the observed data.
2.9. Statistical Analysis
Statistical analysis was performed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA). A p value of <0.05 was considered statistically significant. Pharmacokinetic parameters are reported as geometric mean (min-max), while Tmax is reported as median (min-max). Shapiro–Wilk test was used to verify normal distribution of the pharmacokinetic parameters. Independent t test was performed to analyze statistically significant differences between treatment groups.
3. Results
3.1. Validation of Analytical Methods
The method has good specificity without any peak in chromatogram at retention time of tolfenamic acid. The linearity of working and calibration curves was excellent in the concentration range of 0.04–20 μg/mL with R2 values > 0.999. The LOD was 0.02 μg/mL and the LOQ was 0.04 μg/mL. The extraction procedure was highly efficient and the mean recovery ratio of tolfenamic acid was above 95%. The coefficients of variation for intra- and inter-day were <6.88% and <5.49%, respectively. The intra- and inter-day bias were ±4.15% and ±3.23%, respectively. Overall, the validated approach has demonstrated to be reliable for measuring tolfenamic acid in cats’ plasma using HPLC.
3.2. Pharmacokinetics
The semi-logarithmic plasma concentration–time curve following subcutaneous administration of tolfenamic acid at a dose of 4 mg/kg in cats undergoing ovariohysterectomy with different anesthesia protocols is presented in Figure 1. Tolfenamic acid was detected up to 24 h in both anesthesia groups. Table 1 presents non-compartmental pharmacokinetic parameters. The t1/2λz and MRT0-∞ of tolfenamic acid were longer in cats undergoing ovariohysterectomy that received xylazine–ketamine anesthesia compared to those that received propofol–isoflurane anesthesia. Cats administered propofol–isoflurane exhibited a greater Cmax and a shorter Tmax than those receiving xylazine–ketamine anesthesia (p < 0.05). The AUC, Vdarea/F, and ClT/F were comparable in both anesthesia groups (p > 0.05).
4. Discussion
This study revealed for the first time the pharmacokinetics of tolfenamic acid in cats undergoing ovariohysterectomy under xylazine–ketamine and propofol–isoflurane anesthesia. The study concluded that pharmacokinetic parameters of tolfenamic acid, such as t1/2λz, Cmax, and Tmax, varied depending on the anesthesia protocol.
Overall, glucuronidation metabolism plays a crucial role in the elimination of NSAIDs. The distribution of these medications in the cat varies from that in the dog due to the low capacity of hepatic glucuronidation in the cat [5]. It has been reported that the t1/2λz of NSAIDs, especially those metabolized via glucuronidation reactions, is longer in cats than in dogs [5]. Therefore, there is a significant potential for NSAID toxicity in cats, and using this group of drugs at the appropriate dose and dosing interval is crucial for safety [30,31]. The adverse effects of NSAIDs are particularly related to the gastrointestinal, renal, and hepatic systems [5]. Tolfenamic acid has a lower risk of gastrointestinal damage than many other NSAIDs and is well tolerated at therapeutic doses [32,33,34]. Similarly, oral administration of tolfenamic acid at a dose of 4 mg/kg for 14 days has no adverse effects on renal function in cats [35]. The tolfenamic acid at a dosage of 4 mg/kg for 3 days did not induce any change in diarrhea, vomiting, polyuria, polydipsia, or aggression in the cats [5].
The recommended dose of tolfenamic acid in cats is 4 mg/kg via oral or subcutaneous route [5,14]. Tolfenamic acid was administered subcutaneously at a dosage of 4 mg/kg to cats getting ovariohysterectomy and showed efficacy [15,16]. For ovariohysterectomy procedures in cats, a single dose of NSAID injection is frequently suggested [7]. Preoperative analgesic therapy is recommended because it can either lead to higher plasma and tissue fluid/inflammatory exudate levels or reduce central changes by decreasing the amount of noxious information generated peripherally [36,37]. Therefore, in this study, a single dose of 4 mg/kg tolfenamic acid was administered subcutaneously 30 min before ovariohysterectomy.
Physiological alterations occur in the body due to surgical procedures and anesthetic agents. Surgery causes stress associated with pain, anxiety, cardiovascular instability, and hemodynamic changes, triggering the release of catecholamines, corticosteroids, and cytokines, and in turn initiating a complex series of metabolic events that can last for several days [38]. Anesthetic drugs alter body perfusion, hepatic and metabolic functions, renal functions, and the binding of drugs to plasma proteins [27]. Therefore, surgical procedures may cause differences in the pharmacokinetics of concomitantly used drugs [39]. Ovariohysterectomy surgery altered the disposition of meloxicam and delayed the elimination of carprofen in dogs [36,40].
The t1/2λz is defined as the time it takes for a drug’s concentration in the body to decrease to half of its initial amount [41]. The t1/2λz of tolfenamic acid in cats undergoing ovariohysterectomy was longer in the xylazine–ketamine than in the propofol–isoflurane anesthesia groups. However, the difference between the two anesthetic groups was only 11%, which may be clinically insignificant. The t1/2λz is a parameter derived from both clearance and volume of distribution and is affected by changes in these parameters [42]. The Vdarea/F and ClT/F values were comparable in both anesthesia groups in this study. Since the absorption process will not have an impact, intravenous administration is the most suitable route for determining these two parameters. In this study, since tolfenamic acid was administered solely via the subcutaneous route, variations in absorption across the different anesthesia groups could not be determined. Although these two parameters were similar in both anesthesia groups, the change at t1/2λz may be due to differences in absorption between the anesthesia groups.
The Vdarea/F and ClT/F of tolfenamic acid were similar in cats undergoing ovariohysterectomy under xylazine–ketamine and propofol–isoflurane anesthesia. NSAIDs generally have a limited volume of distribution owing to their significant binding to plasma proteins and their ionized condition in the bloodstream. Fenamate derivatives such as tolfenamic acid have pKa values of approximately 4 and are largely ionized at physiological pH levels (blood = 7.4) [43]. Tolfenamic acid has a high binding to plasma proteins in a variety of animal species, including the dog [21]. Therefore, tolfenamic acid generally has a low volume of distribution [44]. Tolfenamic acid undergoes hepatic metabolism and is eliminated via urine and bile, along with its metabolites [45,46]. It has been reported that it is mostly excreted unchanged in cats [14]. Surgical procedures and anesthetics have been reported to affect cardiac output, tissue perfusion, renal and hepatic blood flow, biotransformation enzyme activities, and plasma protein levels, and this may affect the volume of drug distribution and clearance [27,39,47]. Since there was no control group that did not undergo surgery in this study, the effect of anesthesia protocols on this could not be evaluated. However, in dogs undergoing ovariohysterectomy procedure, the Vd value of meloxicam decreased after IV administration, while the Cl value remained unchanged [40]. The effects of xylazine–ketamine and isoflurane anesthesia on hemodynamics and cardiac function [28], the effects of xylazine–ketamine and ketamine-propofol anesthesia on pulse rate, rectal temperature, and hematobiochemical profile [48], and the effects of medetomidine, xylazine–ketamine–halothane, and propofol on glomerular filtration differed [49]. These results show that the pathophysiological effect differs according to the anesthesia protocol. To our knowledge, there are no studies comparing the pathophysiological effects of xylazine–ketamine and propofol–isoflurane anesthesia protocols; however, since these two anesthesia protocols exhibit similar effects, the Vdarea/F and ClT/F may be similar in these groups.
The Cmax of tolfenamic acid was higher and the Tmax shorter in cats treated with propofol–isoflurane than in animals treated with xylazine–ketamine anesthesia. This shows that tolfenamic acid reaches higher concentrations in the blood in a shorter time after propofol–isoflurane anesthesia. The Cmax indicates the rate and extent of absorption from the injection site of the drug according to the elimination rate [50]. The rate and extent of drug absorption can vary depending on the drug formulation, injection site, and tissue perfusion. Both groups were used with the same formulation of tolfenamic acid, and the injections were administered in the same area. Therefore, changes in these parameters may result particularly from changes in tissue perfusion. During surgical procedures, decreased cardiac output and peripheral perfusion reduce the absorption of drugs from the injection site, which can lead to a decrease in Cmax and a prolongation of Tmax [27]. The effects of anesthetic agents on hemodynamics and cardiac function vary [28]. Xylazine–ketamine anesthesia caused more changes in hemodynamics, cardiac function, and glucose and lipid metabolism compared to isoflurane anesthesia [28]. Heart rate decreased significantly 15 min after xylazine–ketamine anesthesia but was similar to that of isoflurane at 40 min [28]. In this study, the differences in Cmax and Tmax values of tolfenamic acid may be due to differences in hemodynamics and cardiac functions among the anesthesia groups. However, it should be noted that this assessment is merely an interpretation, as the effects of anesthetics on hemodynamics and cardiac functions were not evaluated in this study.
The AUC of tolfenamic acid was similar in cats undergoing ovariohysterectomy under xylazine–ketamine and propofol–isoflurane anesthesia. Tolfenamic acid was detected up to 24 h in both anesthesia groups. AUC represents the total systemic exposure to a drug over time, and systemic clearance and bioavailability play a crucial role in determining AUC (Dose × F/Cl) [51,52]. The similarity of AUC in both anesthesia groups may be due to the relationship between systemic clearance and bioavailability. The fact that the AUC value was similar in both anesthesia groups may indicate that the systemic exposure to tolfenamic acid is similar and that it can be used with two different anesthesia protocols.
To the best of our knowledge, pharmacodynamic studies regarding the dose-concentration relationship of tolfenamic acid in cats are insufficient; therefore, the relationship between plasma concentration and pharmacodynamic efficacy could not be compared. In addition, it has been reported that plasma concentrations of NSAIDs do not reflect their effectiveness [53]. Plasma proteins tend to accumulate in the inflammatory region, and drugs that bind highly to these proteins, such as tolfenamic acid, can accumulate in this region and exert a longer-lasting effect [10]. Therefore, the dose–response relationship of tolfenamic acid in cats undergoing ovariohysterectomy needs to be established.
The findings of this study should be interpreted in light of certain limitations. First, the relatively small number of animals (n = 6) used in each group may limit the broader generalization of the pharmacokinetic results. Second, because there was no non-surgical control group, the absolute effects of the surgical procedure and anesthesia protocols on the pharmacokinetics of tolfenamic acid from a baseline state could not be evaluated. Third, although the alterations in the absorption phase were attributed to hemodynamic changes, specific cardiovascular parameters such as cardiac output or blood pressure were not recorded during the surgeries. Fourth, since NSAIDs—including tolfenamic acid—tend to accumulate in exudate fluid, plasma concentrations may not reflect the therapeutic effect. Therefore, studies characterizing the exudate concentration and pharmacokinetics of tolfenamic acid in cats are needed to evaluate its therapeutic effect. Fifth, although postoperative pain and the recovery process were clinically assessed, no feline pain scoring system was employed. Therefore, the inability to fully rule out confounding effects associated with pain—and the inability to assess the relationship between pain severity and the biomarker—represents a limitation. Additionally, the plasma protein binding ratio and the metabolism of tolfenamic acid were not determined in cats undergoing ovariohysterectomy, which could have provided deeper mechanistic insights. Finally, as this research focused exclusively on pharmacokinetics, the clinical efficacy and dose–response relationship of tolfenamic acid under these anesthetic protocols remain to be evaluated in future studies.
5. Conclusions
This study demonstrates for the first time the pharmacokinetic profile of subcutaneously administered tolfenamic acid in cats undergoing ovariohysterectomy under different anesthesia protocols. Although propofol–isoflurane anesthesia resulted in statistically significant alterations in the absorption phase—specifically a higher peak Cmax and a shorter time to reach peak concentration Tmax—these variations are unlikely to be of clinical significance. The comparable overall systemic exposure (AUC), apparent clearance, and relatively minor difference in the t1/2λz between the groups indicate that tolfenamic acid can be safely and effectively administered at the standard 4 mg/kg dose regardless of the chosen anesthetic regimen, without the need for dosage adjustments. Nevertheless, to fully optimize postoperative pain management in feline practice, future studies should investigate the pharmacodynamic efficacy, dose–response relationships, and target tissue accumulation of tolfenamic acid in surgical patients. Furthermore, this study did not include a group of cats that received either anesthesia regimen without undergoing a surgical procedure. Consequently, it is difficult to distinguish whether the observed alteration in the pharmacokinetics of tolfenamic acid resulted from surgical trauma/stress or the effects of the anesthetic agents. Therefore, further studies incorporating anesthetic control groups are needed.