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Volume 38 Issue 3
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ZHANG Yu, QIN Zhen, SUN Yang, LÜ Jiaguo, WU Fang, LIU Xia. Pharmacokinetics of HMS-01 in rats[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(3): 237-240, 249. doi: 10.12206/j.issn.1006-0111.201907036
Citation: ZHANG Yu, QIN Zhen, SUN Yang, LÜ Jiaguo, WU Fang, LIU Xia. Pharmacokinetics of HMS-01 in rats[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(3): 237-240, 249. doi: 10.12206/j.issn.1006-0111.201907036

Pharmacokinetics of HMS-01 in rats

doi: 10.12206/j.issn.1006-0111.201907036
  • Received Date: 2019-07-09
  • Rev Recd Date: 2020-05-06
  • Available Online: 2020-05-20
  • Publish Date: 2020-05-01
  •   Objective  To study the pharmacokinetics of HMS-01 in rats and provide support for subsequent study.  Methods  A sensitive and specific method for the determination of HMS-01 in plasma and other biological samples was established by LC-MS/MS. The pharmacokinetics of HMS-01 in rats was studied by the established method. The pharmacokinetics of one dose of single intragastric administration and one dose of single intravenous administration in SD rats were studied, and the basic pharmacokinetic parameters were obtained.  Results  After intravenous injection of 1 mg/kg HMS-01, the area under the plasma concentration-time curve AUC0-t of male and female rats was 221 ng·h/ml and 409 ng·h/ml, respectively. The average clearance rates were 4.53 L/h·kg and 2.41 L/h·kg, respectively. The average plasma elimination half-lives were 0.786 h and 1.27 h, and the apparent distribution volume was 5.13 L/kg and 3.82 L/kg, respectively. After intragastric administration of 30 mg/kg HMS-01, the peak time of plasma concentration in rats was 1.17 h, the peak concentration of Cmax was 1 243 ng/ml, and the elimination half-life t1/2 was 2.00 h. The AUC0-t of male and female rats was 2 271 and 8 529 ng·h/ml respectively, and their bioavailability was 34.3% and 69.5% respectively.  Conclusion  The pharmacokinetics of HMS-01 in rats has significant gender differences. It is well absorbed orally, and the bioavailability of HMS-01 in females is much higher than that in males.
  • [1] GANDEVIA S C. Spinal and supraspinal factors in human muscle fatigue[J]. Physiol Rev,2001,81(4):1725-1789.
    [2] BAO L, CAI X X, WANG J B, et al. Anti-fatigue effects of small molecule oligopeptides isolated from Panax ginseng C. A. Meyer in mice[J]. Nutrients,2016,8(12):E807.
    [3] VERMA S K, RAJEEVAN V, JAIN P, et al. Effect of garlic (Allium sativum) oil on exercise tolerance in patients with coronary artery disease[J]. Indian J Physiol Pharmacol,2005,49(1):115-118.
    [4] ØRTENBLAD N, WESTERBLAD H, NIELSEN J. Muscle glycogen stores and fatigue[J]. J Physiol,2013,591(18):4405-4413.
    [5] 赵妍, 张梅, 任爱农, 等. LC-MS/MS分析大鼠口服清清颗粒后血浆中9种成分的药代动力学[J]. 中国实验方剂学杂志, 2017, 23(1):85-90.
    [6] 吴晓霞, 彭娟, 范斌, 等. LC-MS-MS测定黄连解毒汤中3种生物碱在大鼠血清的含量及其药代动力学研究[J]. 中国中药杂志, 2009, 34(10):1276-1280. doi:  10.3321/j.issn:1001-5302.2009.10.023
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Pharmacokinetics of HMS-01 in rats

doi: 10.12206/j.issn.1006-0111.201907036

Abstract:   Objective  To study the pharmacokinetics of HMS-01 in rats and provide support for subsequent study.  Methods  A sensitive and specific method for the determination of HMS-01 in plasma and other biological samples was established by LC-MS/MS. The pharmacokinetics of HMS-01 in rats was studied by the established method. The pharmacokinetics of one dose of single intragastric administration and one dose of single intravenous administration in SD rats were studied, and the basic pharmacokinetic parameters were obtained.  Results  After intravenous injection of 1 mg/kg HMS-01, the area under the plasma concentration-time curve AUC0-t of male and female rats was 221 ng·h/ml and 409 ng·h/ml, respectively. The average clearance rates were 4.53 L/h·kg and 2.41 L/h·kg, respectively. The average plasma elimination half-lives were 0.786 h and 1.27 h, and the apparent distribution volume was 5.13 L/kg and 3.82 L/kg, respectively. After intragastric administration of 30 mg/kg HMS-01, the peak time of plasma concentration in rats was 1.17 h, the peak concentration of Cmax was 1 243 ng/ml, and the elimination half-life t1/2 was 2.00 h. The AUC0-t of male and female rats was 2 271 and 8 529 ng·h/ml respectively, and their bioavailability was 34.3% and 69.5% respectively.  Conclusion  The pharmacokinetics of HMS-01 in rats has significant gender differences. It is well absorbed orally, and the bioavailability of HMS-01 in females is much higher than that in males.

ZHANG Yu, QIN Zhen, SUN Yang, LÜ Jiaguo, WU Fang, LIU Xia. Pharmacokinetics of HMS-01 in rats[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(3): 237-240, 249. doi: 10.12206/j.issn.1006-0111.201907036
Citation: ZHANG Yu, QIN Zhen, SUN Yang, LÜ Jiaguo, WU Fang, LIU Xia. Pharmacokinetics of HMS-01 in rats[J]. Journal of Pharmaceutical Practice and Service, 2020, 38(3): 237-240, 249. doi: 10.12206/j.issn.1006-0111.201907036
  • 疲劳的定义为机体生理过程不能维持其机能在一特定水平上,或不能维持预定的运动强度[1]。由于疲劳的分子机制并不清楚,目前并无真正官方认可的抗疲劳药物。一些天然产物,如人参、红景天、大蒜等,以及营养补充剂,如维生素、矿物质和肌酸等,报道有抵抗肌肉疲劳、提高运动成绩的效应,但是效果缓慢而不显著[2];苯丙胺、莫达非尼、咖啡因等药物虽能快速改善疲劳,但实质为中枢兴奋药,存在成瘾、耐受、改变生物节律等问题[3]。因此,抗疲劳药物的进一步研发,对改善生活质量、提升运动成绩、提高军事作业能力有重要意义。

    组织糖原含量与疲劳的发生密切相关[4]。本课题组通过大规模化合物合成和筛选,获得了一个全新小分子化合物HMS-01,可以通过促进肝脏和肌肉组织糖原的储存,改善疲劳后组织损伤,显著增加肌肉耐力,发挥抗疲劳的作用。为了进一步了解HMS-01的药动学特点以及在组织中的分布,本课题组采用液相色谱-串联质谱(LC-MS/MS)技术[5-6],研究建立灵敏、特异的测定血浆等生物样品中HMS-01浓度的分析方法,并开展HMS-01在大鼠体内的药动学研究,为后续药物研发提供理论依据。

  • Agilent 1290 InfinityⅡ液相色谱仪(Agilent Technologies,美国)、4000 Q-Trap型串联质谱仪(AB Sciex,美国);低温高速离心机(Thermo,德国);XW 80A型涡旋混合器(医大仪器,上海);Mettler AE240十万分之一电子天平(梅特勒-托利多,瑞士);Millipore-Q超纯去离子水净化仪(Millipore,美国)。

  • HMS-01(西安秦申嘉合药物研究有限公司,批号HMS-1-1010-3);罗红霉素(Sigma,CAS:2058-46-0);乙腈(色谱纯,Fisher Chemical);甲酸(色谱纯,Fisher Chemical);其他试剂为市售分析纯。

  • 色谱柱为Gemini C18(50 mm×2.0 mm, 5 μm),流动相:含5 mmol甲酸铵及1 mmol甲酸的水溶液(A)-含1 mmol甲酸的乙腈(B),梯度洗脱,洗脱程序如下:0~2 min,90% A;2~6 min,5% A;6~8 min,90% A。流速:0.35 ml/min,柱温:25 ℃,进样量5μl,运行时间8 min。

  • 采用ESI正离子模式,多反应离子监测模式(MRM)进行二级扫描;动态反应监测的离子对参数:HMS-01 772.5→614.5;罗红霉素(IS)837.5→158.1。离子源参数设置:干燥气温度350 ℃;干燥气流速10 L/min;雾化器压力40 psi;鞘气温度400 ℃;鞘气流速11 L/min;毛细管电压4 000 V;喷嘴电压500 V。HMS-01保留时间为2.834 min,罗红霉素保留时间为2.839 min,色谱图如图1所示。

  • 精密称取HMS-01对照品772.5 mg,置于10 ml容量瓶中,加甲醇溶解并稀释至刻度,摇匀,得质量浓度为77.25 mg/ml对照品储备液。再采用逐级稀释法配置质量浓度为772.5、386.25、193.125、96.563、48.281、24.141、12.070、6.035、3.018、1.509、0.754 ng/ml的系列含对照品血浆溶液;随行质控样品中HMS-01的低、中、高质量浓度分别为3.09、30.9、618 ng/ml。以上溶液置于4 ℃冰箱备用。

  • 取50 μl血浆样品于离心管中,加入200 μl(含41.875 ng/ml的IS内标)乙腈溶液,涡旋60 s,4 ℃条件下3 000×g离心10 min,取上清液进样。

  • 按“2.3”项和“2.4”项中的方法制备标准曲线,平行操作5份,按上述LC-MS/MS条件,连续进样分析,以对照品浓度(X)为横坐标,HMS-01的峰面积与内标的峰面积比值(Y)为纵坐标,进行线性回归,得线性方程:

    Y=0.009 3 X+0.005 08, r = 0.999 3,

    线性范围为0.976~1 000 ng/ml,以信噪比为3或5时测得HMS-01的最低定量限均为976 pg/ml。

  • 按“2.3”项和“2.4”项中的方法制备低、中、高3种浓度的质控样品,平行操作5份,连续3 d,计算实测浓度。如表1所示,精密度用相对标准偏差(RSD)表示,结果日内精密度RSD≤12%,日间精密度RSD≤9%;准确度以相对回收率表示,实测浓度与理论加入浓度的比值即为相对回收率。结果表明日内、日间结果准确度范围在87%~106%。

    浓度(ng/ml)日内日间
    实测浓度(ng/ml)精密度(%)准确度(%)实测浓度(ng/ml)精密度(%)准确度(%)
    3.092.71±0.277.787.92.71±0.277.587.9
    30.931.4±4.811.810232.6±3.68.4106
    618618±465.8100618±465.498.4
  • 按“2.3”项和“2.4”项中的方法制备低、高2种浓度含药血浆,平行操作6份;取空白血浆按“2.4”项下处理,甲醇代替内标溶液,取处理好的空白血浆样品加入相应浓度对照品溶液,使之浓度与待测物的峰面积理论浓度一致,分别制备6份;将待测化合物标准溶液用甲醇稀释,使之与待测物的峰面积理论浓度一致,进样6次。基质效应等于(含基质样品的峰面积)/(80%乙腈溶液的峰面积),提取回收率等于(待测物的峰面积)/(含基质样品的峰面积),考察内标的基质效应和提取回收率操作步骤同上。低、高两种浓度待测物及内标提取回收率均在60%~75%之间,基质效应均在5.6%~6.1%之间,具体结果见表2

    待测物浓度(ng/ml)提取回收率(%)基质效应(%)
    HMS-013.0968±36.1
    61863±45.7
    内标3.3573±26.1
    67073±25.6
  • 按“2.3”项和“2.4”项中的方法制备低、高2种浓度含药血浆,分别考察样品前处理后室温放置24 h,3次冻融循环以及-70 ℃保存30 d的稳定性,测定样品浓度,计算平均值,并计算RSD(%)及相对偏差RE(%),计算公式:RE%=(实测值−真实值)/真实值×100%。结果见表3。测定结果的RE范围为2%~17%,RSD均小于7%,表明样本稳定性良好。

    条件浓度(ng/ml)RSD(%)RE(%)
    室温24 h3.093.716.5
    6181.02.6
    3次冻融循环3.096.74.5
    6180.65.9
    –70 ℃保存30 d3.066.88.7
    6.180.610.1
  • SD大鼠12只,分成2组,每组6只,雌雄各半。给药前禁食12 h,自由饮水。按30 mg/kg的剂量单次灌胃、1 mg /kg的剂量单次静注给药。灌胃给药的,于给药前和给药后5、10、20、30 min和1、1.5、2、4、6、8、10、12、24 h分别眼眶采血约100 µl;静注给药的,于给药前和给药后3、8、15、30 min和1、2、3、4、6、8、10、12、24 h分别眼眶采血约100 µl。血液用1%肝素抗凝,8 000×g离心5 min,分离血浆。–70 ℃保存待测。采血过程冰浴避光,以防止光对药物稳定性产生影响。

    大鼠单次静注给予1 mg/kg HMS-01后的药动学参数(非房室模型)总结于表4。大鼠静脉注射1 mg/kg HMS-01后,雄、雌大鼠药动学参数血浆浓度-时间曲线下面积(AUC)、平均清除率(CL)显示有极显著性差异(P<0.01),药动学参数血浆消除半衰期(t1/2)、表观分布容积(V)均呈显著性差异(P<0.05)。雄性大鼠AUC0-t为221 ng·h/ml,CL为4.53 L/h·kg,为大鼠肝脏血流量(约3.3 L/h·kg)的137%,体内清除快, t1/2约为0.786 h,V为5.13 L/kg;雌性大鼠AUC0-t为409 ng·h/ml,CL为2.41 L/h·kg,为大鼠肝脏血流量的73%,体内清除较快, t1/2约为1.27 h,V为3.82 L/kg。

    性别AUC0-t (ng·h/ml)AUC0-∞ (ng·h/ml)MRT0-∞ (t/h)t1/2z (t/h)CLz (L/h·kg)Vz (L/kg)cmax (ng/ml)
    雄性221±12.6221±12.40.776±0.0220.786±0.0394.53±0.2525.13±0.388491±112
    雌性409±23.3**416±21.0**1.270±0.1151.090±0.141*2.41±0.120**3.82±0.666*571±55.1
    合计315±105319±1081.030±0.2820.940±0.1933.47±1.174.47±0.871531±90.3

      大鼠单次灌胃给予30 mg/kg HMS-01后的药动学参数(非房室模型)总结见表5。灌胃给予30 mg/kg HMS-01后,雄、雌大鼠药动学参数AUC显示有极显著性差异(P<0.01),药动学参数CLF显示有显著性差异(P<0.05)。在大鼠体内血浆浓度达峰时间tmax为1.17 h,达峰浓度cmax为1 243 ng/ml,消除半衰期t1/2为2.00 h。雄、雌大鼠AUC0-t分别为2 271和8 529 ng·h/ml,生物利用度分别为34.3%和69.5%。

    性别AUC0-t (ng·h/ml)AUC0-∞ (ng·h/ml)MRT0-∞ (t/h)t1/2z (t/h)tmax (t/h)CLz/F (L/h·kg)Vz/F (L/kg)cmax (ng/ml)F(%)
    雄性2 271±6662 279±6672.58±0.1561.43±0.1301.17±0.28914.00±4.3128.5±7.04729±26334.3±10.1
    雌性8 529±1 920**9 071±1 529**4.79±1.392.58±1.0601.17±0.2893.38±0.629*13.1±7.781757±58469.5±15.6*
    合计5 400±3 6615 675±3 8673.68±1.502.00±0.9241.17±0.2588.69±6.4420.8±10.71 243±69451.9±22.6
  • HMS-01在大鼠体内的药动学过程存在显著的性别差异。口服吸收的比较,雌性大鼠的生物利用度远高于雄性,体内的清除速率方面,雌性比雄性慢,与此同时,HMS-01在雌性体内的半衰期也更长。存在性别差异的原因有待进一步深入研究。本实验采用的分析方法的特异性、灵敏性、准确性、精密度及稳定性均满足定量分析的要求。鉴于HMS-01在大鼠血浆中不稳定的情况,在做动物实验时,课题组将采取以下措施:全血采集后立刻在4 ℃下离心2 min获取血浆,随即立刻取50 µl血浆样品加入至200 µl含内标的乙腈中,从而阻断血浆中的水解酶对化合物进行水解。

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