青蒿素脂质纳米粒的制备及药动学评价

doi:10.5246/jcps.2017.03.018

摘要/Abstract

摘要：

青蒿素(ART)是一种广泛用于治疗严重疟疾和脑型疟疾的药物。但因其口服生物利用度低影响了药物疗效。本研究将ART搭载于的纳米结构脂质载体系统(NLC), 以期改善药物的药动学特征。采用高压乳匀法, 并运用正交设计优化制备青蒿素脂质纳米粒(ART-NLC)。得到的ART-NLC粒径, zeta电位, 包封率, 载药量分别为(53.06±2.11) nm, (−28.7±3.59) mV, 73.9%±0.5%, 11.23%±0.37%。ART-NLC在体外呈现缓释特性, 且对人体红细胞未发现显著的溶血性。采用液相色谱串联质谱法(LC-MS/MS)测定经尾静脉注射或腹腔注射ART-NLC后, 大鼠的药物动力学参数, 以ART溶液剂作为对照组。大鼠经尾静脉注射给药后, ART-NLC组的AUC0–∞ ((707.45±145.65) ng·h/mL)明显比ART组((368.98±139.58) ng·h/mL)大。ART-NLC组的MRT ((3.38±0.46) h)较ART组((1.39±0.61) h)有所延长。大鼠经腹腔注射给药后, 得到相同结果。即ART-NLC组的AUC0–∞ ((1233.06 ±235.57) ng·h/mL)和MRT ((4.97±0.69) h)都比ART组的AUC0–∞ ((871.17±234.03) ng·h/mL)和MRT ((1.75±0.31) h)大。大鼠经尾静脉和腹腔注射给药后, ART-NLC组的AUC0–∞ (P<0.05)和MRT (P<0.001)较ART组均显著性增加。

关键词: 青蒿素, 纳米结构脂质载体, 药代动力学, 液相色谱串联质谱

Abstract:

Artemisinin (ART) is a widely used active drug for malaria, including severe and cerebral malaria. However, its therapeutic efficacy is affected by its lower bioavailability. In the present study, nanostructured lipid carriers (NLCs) were proposed as carrier of ART to improve pharmacokinetic properties of the drug. ART-NLC was prepared by high-pressure homogenization based on orthogonal design. The particle size, zeta potential, encapsulation efficiency (EE) and percentage of drug loading (DL) of ART-NLC were (53.06±2.11) nm, (–28.7±3.59) mV, 73.9%±0.5% and 11.23%±0.37%, respectively. ART-NLC showed the sustained release characteristics and scarcely the hemolysis effect on human red blood cells. The pharmacokinetics of ART-NLC for rats after tail intravenous injection (i.v) or intraperitoneal injection (i.p) were investigated by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). And ART solution was designed as control preparation. For rats of i.v groups, the AUC0–∞ ((707.45±145.65) ng·h/mL) of ART-NLC were significantly bigger than that of ART ((368.98±139.58) ng·h/mL). The MRT ((3.38±0.46) h) of ART-NLC was longer than that of ART ((1.39±0.61) h). And similar results were observed for rats of i.p groups. The AUC0–∞ ((1233.06±235.57) ng·h/mL) and MRT ((4.97±0.69) h) of ART-NLC were both bigger than those of ART, which were (871.17±234.03) ng·h/mL) and (1.75±0.31) h), respectively. Compared with ART, ART-NLC showed a significant increase in AUC0–∞ (P<0.05) and MRT (P<0.001) for both i.p and tail i.v administrations.

Key words: Artemisinin, Nanostructured lipid carriers, Pharmacokinetics, LC-MS/MS

中图分类号:

R969.1

Supporting:

Method validation

1. Chromatography specificity

The specificity of the method was evaluated by analyzing six different blank samples of rat plasma (Fig. 5A); a blank rat plasma spiked with ART at LLOQ (Fig. 5B); a blank rat plasma spiked with IS (Fig. 5C); a blank rat plasma spiked with ART (Fig. 5D); a blank rat plasma spiked with ART and ARM (Fig. 5E); a rat plasma sample after injection of ART (Fig. 5F). It could be inferred from Fig. 5 that no interfering endogenous substances were detected at the retention time of ART and IS.

Figure 1. Representative MRM chromatograms.

2. Linearity and LLOQ

The calibration curve could be constructed by plotting the peak-area ratios (y) of ART to IS versus the concentrations (x) of ART using weighed least squares linear regression (1/x²). The satisfying equation of calibration curve was obtained: y = 2.25 + 0.223 x, (r = 0.9990). The LLOQ of ART in rat plasma (n = 5) was established at 2.0 ng/mL equal to 10-fold of the ratio of signal-to-noise approximately (S/N =10).

3. Accuracy and precision

The accuracy and precision of the method were assessed by determining QC samples (5, 250 and 320 ng/mL) using six replicated preparations of plasma samples on three separated days. The precision and the accuracy were expressed using relative standard deviation (RSD%) and the relative deviation (RE%), respectively. It was acceptable when the values of RE (%) and RSD (%) were less than 15% for the medium and high concentration, and 20% for the low concentration. The method was proved to be highly accurate with 97.6%-109.3% deviation from the nominal values, and highly precise with within-assay precision <4.5%, between-assay precision <12.0% at each concentration of QC sample tested.

4. Matrix effect and recovery

The matrix effect was evaluated by comparing the peak areas obtained from ART spiked in the extracted blank plasma with those from ART solutions at concentrations of 5, 250 and 320 ng/mL, respectively. The way of assessing matrix effect of IS at 200 ng/mL was the same as ART. The matrix effect could be considered negligible when the ratios ranged from 85% to 115%. The extraction recovery of ART was investigated by comparing the peak areas of ART from QC samples with those from standard solutions without extration procedure at the same nominal concentrations. The recovery of IS was evaluated in a similar way at a work concentration of 200 ng/mL. The samples were performed in triplicate.

The mean values of matrix effects of ART and IS were 89.63%±1.34% and 91.19%±3.98%, respectively, indicating that the ionizations of ART and IS were not affected by the matrix components in rat plasma. The mean values of recovery of ART and IS (89.29%±1.47% and 88.43%±3.11%, respectively) were all within the acceptable limits.

5. Stability

The stability of ART was investigated by keeping the QC samples under all experienced conditions, including freezing/thawing over three cycles, autosampler vials at ambient temperature for 24 h, the freezer storage of ART in plasma at -20 °C for 4 weeks. All the QC samples for stability assessment were analyzed in triplicate. The results in Table 4 demonstrated that the plasma samples were stable under the above conditions.

Table 1. Stability of ART in rat plasma (n = 6).

Storage conditions	Concentration
Storage conditions	Add (ng/mL)	Found (ng/mL)	RE (%)	RSD (%)
Three freeze/thaw cycles	5	5.21	104.2	6.9
	250	245.58	98.2	1.8
	320	315.29	98.5	7.2
-20 °C for 4 weeks	5	5.61	112.2	3.4
	250	262.12	104.8	4.8
	320	289.71	90.5	6.6
Treated samples at room temperature for 24 h	5	5.28	105.5	6.2
	250	258.89	103.6	3.6
	320	310.30	96.9	8.1

代华灵, 高伟祺, 张国顺, 王锐利, 张淑秋. 青蒿素脂质纳米粒的制备及药动学评价[J]. 中国药学(英文版), 2017, 26(3): 180-186.

Hualing Dai, Weiqi Gao, Guoshun Zhang, Ruili Wang, Shuqiu Zhang. Preparation of the nanostructured lipid carriers of artemisinin and its pharmacokinetic evaluation[J]. Journal of Chinese Pharmaceutical Sciences, 2017, 26(3): 180-186.

参考文献

[1] Liu, J.M.; Ni, M.Y.; Fan, J.F. Chin. Sci Bull. 1979, 37, 129-141.

[2] Fan, R.; Xie, H.; Yang, J. Acta Acad. Med. CPAPF. 2006, 15, 199-201.

[3] Aweeka, F.T.; German, P.I. Clin. Pharmacokinet. 2008, 47, 91-102.

[4] LeThi, D.T.; Le, N.H.; Nguyen, C.H. Drug Metab. Pharmacokinet. 2008, 23, 158-164.

[5] Davis, T.M.; Karunajeewa, H.A.; Ilett, K.F. Med. J. Aust. 2005, 182, 181-185.

[6] Aweeka, F.T.; German, P.I. Clin. Pharmacokinet. 2008, 47, 91-102.

[7] Yan, H.X.; Xing, J.; Zhang, L.F.; Zhang, S.Q. Asian J. Pharmacodyn. Pharmacokin. 2008, 8, 37-42.

[8] Zhang, X.X.; Gan, Y.; Yang, X.G. Acta Pharm. Sin. 2008, 43, 91-96.

[9] Ren, G.L.; Liu, D.; Guo, W.L. Drug Deliv. 2016, 23, 1272-1281.

[10] Hu, F.Q.; Jiang, S.P.; Du, Y.Z. Int. J. Pharm. 2006, 314, 83-89.

[11] Jia, L.J.; Shen, J.Y.; Zhang, D.R.; Duan, C.X.; Liu, G.P.; Zheng, D.D.;Tian X.N.; Liu, Y.; Zhang, Q. Int. J. Biol. Macromol. 2012, 50, 523-529.

[12] Saliba, K.J.; Martin, R.E.; Staines, H.M.; Kirk, K. Oxford: Isis Medical Media. 1999, 137-148.

[13] Kirk, K. Physiol. Rev. 2001, 81, 495-537.

[14] Abraham, D.J. Burger’s Medicinal Chemistry and Drug Discovery. Vol 2. 6th. New York: John Wiley Sons Inc. 2003.

[15] Müller, R.H.; Luck, M.; Kreuter, J. United States Patent, 6288040, 2001.

[16] Müller, R.H.; Mader, K.; Lippacher, A. PCT application PCT/EP 0004565, 2000.

[17] Zhao, J.H.; Piao, X.H.; Shi, X.Q. Molecules. 2016, 21, 1549-1560.

[18] Ren, W.; Zhang, S.; Zhong, T. J. Chin. Pharm. Sci. 2016, 8, 570-576.

[19] Jenning, V.; Thunemann, A.F.; Gohla, S.H. Int. J. Pharm. 2000, 199, 167-177.

[20] Chinese pharmacopoeia, Guidance for Industry: Bioanalytical Method Validation, 2015.

[21] Gordi, T.; Huong, D.X.; Hai, T.N.; Nieu, N.T.; Ashton, M. Antimicrob. Agents. Chemother. 2002, 46, 1026-1031.

[22] Long, D.H.; Gong, T.; Zhang, Z.R.; Ding, R.; Fu, Y. Acta Pharm. Sin. B. 2016, 6, 329-335.

[23] Xing, J.; Yan, H.X.; Zhang, S.Q.; Ren, G.L.; Gao, Y.H. Rapid Commun. Mass Spectrom. 2006, 20, 1463-1468.

[24] Xie, X.; Zhou, R.; Zhou, P.J. J. Chin. Pharm. Sci. 2016, 9, 676-683.

[25] Zhai, Q.Q.; Pang, J.; Li, G.Q.; Li, C.R.; Yang, X.Y. Acta Pharm. Sin. B. 2015, 5, 467-472.