COVID-19潜在治疗药物洛匹那韦/利托那韦和阿比多尔在大鼠体内的药代动力学相互作用

Pharmacokinetic interactions between the potential COVID-19 treatment drugs lopinavir/ritonavir and arbidol in rats COVID-19潜在治疗药物洛匹那韦/利托那韦和阿比多尔在大鼠体内的药代动力学相互作用 1 first-author 2 1 1 1 1 1 corresp Saiping JIANG, j5145@zju.edu.cn Saiping JIANG, j5145@zju.edu.cn j5145@zju.edu.cn https://orcid.org/0000-0001-7720-2725 Department of Clinical Pharmacy, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou310006, China Department of Clinical Pharmacy, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou310006, China Public Service Platform for the Evaluation of Innovative Drug Property, Hangzhou311121, China Public Service Platform for the Evaluation of Innovative Drug Property, Hangzhou311121, China 目的 2 研究抗病毒药物洛匹那韦/利托那韦和阿比多尔在大鼠体内的药代动力学相互作用。 创新点 2 洛匹那韦/利托那韦联合阿比多尔作为国内多家医疗单位治疗COVID-19患者的基础抗病毒方案，两者的药物相互作用未有研究报道，本论文通过开展体内药动学实验，探索合用后药物体内暴露量的改变，为该方案的临床治疗有效性和安全性提供保障。 方法 2 建立大鼠洛匹那韦/利托那韦与阿比多尔药动学相互作用模型。将大鼠分为3组（阿比多尔组、洛匹那韦/利托那韦组及联用组）。洛匹那韦/利托那韦和阿比多尔分别以50mg/kg/12.5 mg/kg和25 mg/kg灌胃给药。采用液相色谱串联质谱（LC-MS/MS）法测定洛匹那韦和阿比多尔的浓度。 结论 2 洛匹那韦/利托那韦联合阿比多尔治疗后，阿比多尔的药时曲线下面积（AUC）和洛匹那韦的峰浓度（ C max ）显著升高。这两种药物的相互作用可能会影响联合治疗的安全性。因此，洛匹那韦/利托那韦与阿比多尔同时给药时，建议适当调整给药剂量，阿比多尔的剂量可能需要减半。 药代动力学 COVID-19 相互作用 洛匹那韦/利托那韦 阿比多尔 The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occasioned worldwide alarm. Globally, the number of reported confirmed cases has exceeded 84.3 million as of this writing (January 2, 2021). Since there are no targeted therapies for COVID-19, the current focus is the repurposing of drugs approved for other uses. In some clinical trials, antiviral drugs such as remdesivir ( Grein et al., 2020 Grein et al., 2020 ), lopinavir/ritonavir (LPV/r) ( Cao et al., 2020 Cao et al., 2020 ), chloroquine ( Gao et al., 2020 Gao et al., 2020 ), hydroxychloroquine ( Gautret et al., 2020 Gautret et al., 2020 ), arbidol ( Wang et al., 2020 Wang et al., 2020 ), and favipiravir ( Cai et al., 2020b Cai et al., 2020b ) have shown efficacy in COVID-19 patients. LPV/r combined with arbidol, which is the basic regimen in some regional hospitals in China including Zhejiiang Province, has shown antiviral effects in COVID-19 patients ( Guo et al., 2020 Guo et al., 2020 ; Xu et al., 2020 Xu et al., 2020 ). A retrospective cohort study also reported that this combination therapy showed better efficacy than LPV/r alone for the treatment of COVID-19 patients ( Deng et al., 2020 Deng et al., 2020 ). According to the epidemiological studies, 14%‒53% of COVID-19 patients showed abnormal levels of serum aminotransferases during disease progression, which could have been caused by viral infection of liver cells or drug-induced hepatotoxicity ( Zhang et al., 2020 Zhang et al., 2020 ). There was a significantly higher proportion of patients with abnormal liver function after treatment with LPV/r ( Cai et al., 2020a Cai et al., 2020a ). As arbidol produced similar adverse reactions, the combination of these two drugs may increase the risk of liver injury. In our retrospective observational study, 76 of 131 adult COVID-19 patients developed liver injury, and the combination of LPV/r with arbidol was a risk factor for liver injury in non-critical COVID-19 patients ( Jiang et al., 2020 Jiang et al., 2020 ). In the LPV/r regimen, lopinavir is combined with a low dose of ritonavir, which functions as a protease inhibitor. The bioavailability of lopinavir is relatively poor, characterized by rapid and extensive metabolism. The sub-therapeutic dose of ritonavir is used as a potent cytochrome P450 3A4 (CYP3A4) inhibitor to suppress metabolism of lopinavir and boost its plasma concentration, thus increasing its efficacy. Both lopinavir and ritonavir have shown hepatotoxicity in the clinic, producing moderate to severe elevations of serum aminotransferase levels in 3%‒10% and 15% of patients, respectively ( LiverTox, 2012 LiverTox, 2012 ). Arbidol may also elevate serum aminotransferases, but the exact proportion of patients that occurs has not been reported. Given that CYP3A4 is a major isoenzyme involved in the metabolism of arbidol, interactions between LPV/r and arbidol, leading to increased exposure to LPV/r or arbidol, may be expected. Therefore, this interaction may imply a higher risk of hepatotoxicity. Taking these facts into consideration, we performed a study on the pharmacokinetic interactions between LPV/r and arbidol in order to provide some initial guidelines for combined therapy with these two drugs. We established a rat model to investigate the pharmacokinetic interactions between LPV/r and arbidol. Rats were divided into three groups (arbidol, LPV/r, and combination), each consisting of three male and three female rats. Arbidol was administered by gavage at a dose of 25 mg/kg, and LPV/r was composed of 50 mg/kg lopinavir and 12.5 mg/kg ritonavir. The concentrations of arbidol and lopinavir were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As shown in Table 1 Table 1 and Fig. 1 Fig. 1 , when co-administered with LPV/r, the maximum concentration ( C max ) of arbidol increased from (129.8±99.0) to (221.8±112.1) ng/mL ( P > 0.05), and the area under the plasma concentration‒time curve from time zero to infinity (AUC 0–∞ ) increased from (705.6±258.5) to (1250.3±255.5) h·ng/mL ( P < 0.01). The pharmacokinetics of arbidol differed between male and female animals. In the arbidol group, the mean C max and AUC 0–∞ of arbidol were 3.0 and 1.8 times higher in female rats than in male rats, respectively. In the combination group, the mean C max and AUC 0–∞ of arbidol were 2.6 and 1.2 times higher in female rats than in male rats, respectively. Data are expressed as mean±standard deviation (SD). ** P < 0.01, compared with those in the combination group. LPV/r: lopinavir/ritonavir; t 1/2 : half-life; t max : time to maximum concentration; C max : maximum concentration; AUC 0－ t : area under the plasma concentration‒time curve from time zero to the last quantifiable time point; AUC 0－∞ : area under the plasma concentration‒time curve from time zero to infinity. Group t 1/2 (h) t max (h) C max (ng/mL) AUC 0－ t (h·ng/mL) AUC 0－∞ (h·ng/mL) Arbidol  Male ( n =3) 2.7±0.4 0.6±0.1 65.2±16.8 479.0±122.6 497.3±116.8  Female ( n =3) 4.2±1.5 0.5±0.0 194.3±108.1 872.1±166.5 913.9±152.4  Total ( n =6) 3.5±1.3 0.5±0.1 129.8±99.0 675.6±251.9 ** 705.6±258.5 ** Combination  Male ( n =3) 2.7±0.0 0.7±0.1 124.3±29.3 1133.9±206.3 1141.0±207.1  Female ( n =3) 3.6±0.6 0.5±0.0 319.4±44.8 1326.2±267.9 1359.7±290.7  Total ( n =6) 3.2±0.6 0.6±0.1 221.8±112.1 1230.0±238.4 1250.3±255.5 1;2;3; As shown in Table 2 Table 2 and Fig. 2 Fig. 2 , when co-administered with arbidol, the C max of lopinavir increased from (2336.4±504.7) to (3500.6±749.6) ng/mL ( P < 0.05), and the AUC 0–∞ increased from (27 985.1±11 314.6) to (37 101.8±10 083.6) h·ng/mL ( P > 0.05). The pharmacokinetic parameters of lopinavir were also influenced by the sex of the experimental animals. In the LPV/r and combination groups, the mean AUC 0–∞ of lopinavir was 1.9 and 1.4 times higher in female rats than in male rats, respectively, and the time to maximum concentration ( t max ) was also longer in female rats than in male rats. Data are expressed as mean±standard deviation (SD). * P < 0.05, compared with those in the combination group. LPV/r: lopinavir/ritonavir; t 1/2 : half-life; t max : time to maximum concentration; C max : maximum concentration; AUC 0－ t : area under the curve from time zero to the last quantifiable time point; AUC 0－∞ : area under the curve from time zero to infinite. Group t 1/2 (h) t max (h) C max (ng/mL) AUC 0－ t (h·ng/mL) AUC 0－∞ (h·ng/mL) LPV/r  Male ( n =3) 1.5±0.5 6.0±2.0 2098.0±434.8 18 142.9±3955.2 19 042.7±4621.6  Female ( n =3) 3.5±1.8 10.0±2.0 2574.8±526.5 33 196.4±12 906.2 36 927.5±7667.8  Total ( n =6) 2.5±1.6 8.0±2.8 2336.4±504.7 * 25 669.6±11 868.8 27 985.1±11 314.6 Combination  Male ( n =3) 2.0±0.3 6.3±0.6 3565.8±943.3 28 453.9±6323.4 30 438.2±7227.4  Female ( n =3) 1.9±0.4 9.7±0.6 3435.4±708.7 43 657.0±8424.1 43 765.4±8291.4  Total ( n =6) 2.0±0.4 8.0±1.9 3500.6±749.6 36 055.4±10 664.0 37 101.8±10 083.6 1;2;3; Combined antiviral therapy may be an effective approach for curing patients infected with SARS-CoV-2. LPV/r in combination with arbidol is the mainstay of treatment in Zhejiang Province. However, not enough attention has been paid to the safety of these combination drug therapies. Based on clinical observations, the combination of these two drugs may increase the risk of liver injury. Due to their metabolic characteristics, potential drug–drug interactions must be taken into account. Our study is the first to evaluate the pharmacokinetic interactions between LPV/r and arbidol in rats. When combined with LPV/r, exposure to arbidol increased acutely by approximately two times. Meanwhile, the C max of lopinavir increased significantly (1.5 times). Since the pharmacokinetic profile of arbidol is influenced by CYP3A4, one can deduce that drug–drug interactions will change its plasma concentrations. In our study, given the role played by CYP3A4 inhibition, LPV/r increased the bioavailability of arbidol, while arbidol had no obvious effect on the AUC of lopinavir in the presence of ritonavir. A significant, albeit accidental, finding of our study was that the pharmacokinetic profiles of arbidol and lopinavir differed depending on the gender of the experimental animals. Since arbidol was more sensitive to this gender effect, this may influence its safety and efficacy. Gender-based differences in CYP3A expression in rats are likely to explain these observations. This pharmacokinetic characteristic of aribidol has never been reported before. In the case of lopinavir, several clinical studies with patients did not show obvious differences in plasma concentrations between sexes ( Ofotokun et al., 2007 Ofotokun et al., 2007 ; Umeh et al., 2011 Umeh et al., 2011 ). In contrast, Jullien et al. (2006) Jullien et al. (2006) reported that clearance/bioavailability (CL/F) of lopinavir was gender-related, with a 39% increase observed in males compared with females. We acknowledge that our study has some limitations. Firstly, both LPV/r and arbidol were given in a single dose. In clinical practice, arbidol is administered three times a day and LPV/r twice a day. Hence, multiple doses may cause more accumulation of arbidol and lopinavir, and this cannot be ignored. Secondly, this study involved animal experiments and our conclusions have yet to be confirmed in clinical trials. Thirdly, the association between increased arbidol exposure, lopinavir C max , and exacerbated drug-induced liver injury should be studied further. Finally, only three rats were used to compare the pharmacokinetic differences between males and females, which is not sufficient to produce statistically significant results. Therefore, the findings reported here should be considered preliminary. In summary, combined administration of LPV/r and arbidol produced a significant increase in the AUC of arbidol and the C max of lopinavir. The interaction between these two drugs may affect the safety of the combined treatment. Therefore, appropriate dose adjustment is recommended when LPV/r and arbidol are administered concurrently, and the dose of arbidol may need to be halved. The results obtained in this study may be useful to predict interactions between LPV/r and other medications metabolized by CYP3A4. References Cai QX , Huang DL , Yu H , et al. , 2020a . COVID-19: abnormal liver function tests . J Hepatol , 73 ( 3 ): 566 - 574 . https://doi.org/10.1016/j.jhep.2020.04.006 https://doi.org/10.1016/j.jhep.2020.04.006 COVID-19: abnormal liver function tests Cai QX , Yang MH , Liu DJ , et al. , 2020b . Experimental treatment with favipiravir for COVID-19: an open-label control study . Engineering (Beijing) , 6 ( 10 ): 1192 - 1198 . https://doi.org/10.1016/j.eng.2020.03.007 https://doi.org/10.1016/j.eng.2020.03.007 Experimental treatment with favipiravir for COVID-19: an open-label control study Cao B , Wang YM , Wen DN , et al. , 2020 . A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19 . N Engl J Med , 382 ( 19 ): 1787 - 1799 . https://doi.org/10.1056/NEJMoa2001282 https://doi.org/10.1056/NEJMoa2001282 A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19 Deng LS , Li CN , Zeng Q , et al. , 2020 . Arbidol combined with LPV/r versus LPV/r alone against corona virus disease 2019: a retrospective cohort study . J Infect , 81 ( 1 ): E1 - E5 . https://doi.org/10.1016/j.jinf.2020.03.002 https://doi.org/10.1016/j.jinf.2020.03.002 Arbidol combined with LPV/r versus LPV/r alone against corona virus disease 2019: a retrospective cohort study Gao JJ , Tian ZX , Yang X , 2020 . Breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies . Biosci Trends , 14 ( 1 ): 72 - 73 . https://doi.org/10.5582/bst.2020.01047 https://doi.org/10.5582/bst.2020.01047 Breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies Gautret P , Lagier JC , Parola P , et al. , 2020 . Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial . Int J Antimicrob Agents , 56 ( 1 ): 105949 . https://doi.org/10.1016/j.ijantimicag.2020.105949 https://doi.org/10.1016/j.ijantimicag.2020.105949 Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial Grein J , Ohmagari N , Shin D , et al. , 2020 . Compassionate use of remdesivir for patients with severe Covid-19 . N Engl J Med , 382 ( 24 ): 2327 - 2336 . https://doi.org/10.1056/NEJMoa2007016 https://doi.org/10.1056/NEJMoa2007016 Compassionate use of remdesivir for patients with severe Covid-19 Guo YZ , Xu KJ , Li YT , et al. , 2020 . Safety of protease inhibitors and Arbidol for SARS-CoV-2 pneumonia in Zhejiang Province, China . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 21 ( 12 ): 948 - 954 . https://doi.org/10.1631/jzus.B2000204 https://doi.org/10.1631/jzus.B2000204 Safety of protease inhibitors and Arbidol for SARS-CoV-2 pneumonia in Zhejiang Province, China Jiang SP , Wang RR , Li L , et al. , 2020 . Liver injury in critically ill and non-critically ill COVID-19 patients: a multicenter, retrospective, observational study . Front Med (Lausanne) , 7 : 347 . https://doi.org/10.3389/fmed.2020.00347 https://doi.org/10.3389/fmed.2020.00347 Liver injury in critically ill and non-critically ill COVID-19 patients: a multicenter, retrospective, observational study Jullien V , Urien S , Hirt D , et al. , 2006 . Population analysis of weight-, age-, and sex-related differences in the pharmacokinetics of lopinavir in children from birth to 18 years . Antimicrob Agents Chemother , 50 ( 11 ): 3548 - 3555 . https://doi.org/10.1128/AAC.00943-05 https://doi.org/10.1128/AAC.00943-05 Population analysis of weight-, age-, and sex-related differences in the pharmacokinetics of lopinavir in children from birth to 18 years LiverTox , 2012 . Clinical and research information on drug-induced liver injury: protease inhibitors (HIV) . Bethesda (MD) : National Institute of Diabetes and Digestive and Kidney Diseases . https://www.ncbi.nlm.nih.gov/books/NBK548893 https://www.ncbi.nlm.nih.gov/books/NBK548893 Clinical and research information on drug-induced liver injury: protease inhibitors (HIV) Ofotokun I , Chuck SK , Binongo JN , et al. , 2007 . Lopinavir/ritonavir pharmacokinetic profile: impact of sex and other covariates following a change from twice-daily to once-daily therapy . J Clin Pharmacol , 47 ( 8 ): 970 - 977 . https://doi.org/10.1177/0091270007302564 https://doi.org/10.1177/0091270007302564 Lopinavir/ritonavir pharmacokinetic profile: impact of sex and other covariates following a change from twice-daily to once-daily therapy Umeh OC , Currier JS , Park JG , et al. , 2011 . Sex differences in lopinavir and ritonavir pharmacokinetics among HIV-infected women and men . J Clin Pharmacol , 51 ( 12 ): 1665 - 1673 . https://doi.org/10.1177/0091270010388650 https://doi.org/10.1177/0091270010388650 Sex differences in lopinavir and ritonavir pharmacokinetics among HIV-infected women and men Wang ZL , Yang BH , Li QW , et al. , 2020 . Clinical features of 69 cases with coronavirus disease 2019 in Wuhan, China . Clin Infect Dis , 71 ( 15 ): 769 - 777 . https://doi.org/10.1093/cid/ciaa272 https://doi.org/10.1093/cid/ciaa272 Clinical features of 69 cases with coronavirus disease 2019 in Wuhan, China Xu KJ , Cai HL , Shen YH , et al. , 2020 . Management of COVID-19: the Zhejiang experience . J Zhejiang Univ (Med Sci) , 49 ( 2 ): 147 - 157 (in Chinese) . https://doi.org/10.3785/j.issn.1008-9292.2020.02.02 https://doi.org/10.3785/j.issn.1008-9292.2020.02.02 Management of COVID-19: the Zhejiang experience Zhang C , Shi L , Wang FS , 2020 . Liver injury in COVID-19: management and challenges . Lancet Gastroenterol Hepatol , 5 ( 5 ): 428 - 430 . https://doi.org/10.1016/S2468-1253(20)30057-1 https://doi.org/10.1016/S2468-1253(20)30057-1 Liver injury in COVID-19: management and challenges Yunzhen HU, Saiping JIANG, and Xiaoyang LU conceived and designed this study. Minjuan ZUO and Xiaojuan WANG carried out the experiments. Yunzhen HU performed data analysis and drafted the manuscript. Rongrong WANG and Lu LI made critical contribution to the discussion. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work is supported by the National Natural Science Foundation of China (No. 81703612). 10.1631/jzus.B2000728 1673-1581（2021）07-0599-04 2021-01-03 2021-02-21 2021-07-15 2022-05-09 2021-05-12 © Zhejiang University Press 2021 Yunzhen HU, Minjuan ZUO, Xiaojuan WANG, Rongrong WANG, Lu LI, Xiaoyang LU, and Saiping JIANG declare that they have no conflict of interest. The Research Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China) approved the protocol followed in this study. All institutional and national guidelines for the care and use of laboratory animals were followed. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 7 22