Jiaojiao Cong1, Yiling Ruan2, Qinglin Lyu3, Xiaohui Qin4, Xinming Qi5, Wenyuan Liu6, Lifeng Kang7, Junying Zhang8, Chunyong Wu9. 1. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China; China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China. Electronic address: congjiaojiaohenan@163.com. 2. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China; China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China. Electronic address: ryljessica@163.com. 3. China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China. Electronic address: lyu@cpu.edu.cn. 4. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China; China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China. Electronic address: qxh_310@163.com. 5. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No 501 Haike Road, Shanghai, 201203, China. Electronic address: qxmng77777@gmail.com. 6. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China; China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China. Electronic address: liuwenyuan8506@163.com. 7. School of Pharmacy, University of Sydney, Pharmacy and Bank Building A15, NSW 2006, Australia. Electronic address: lkang@alumni.nus.edu.sg. 8. China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China. Electronic address: cpuzjy@cpu.edu.cn. 9. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China; China Pharmaceutical University, No 24 Tongjia Road, Nanjing, 210009, China. Electronic address: cywu@cpu.edu.cn.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: The herbs of Aconitum are the essential Traditional Chinese medicine and have played an indispensable role in many Asian countries for thousands of years to treat critical illnesses, and chronic, stubborn diseases. However, Aconitum may induce severe neurotoxicity and even death. So far the mechanism of Aconitum penetrating the blood-brain barrier (BBB) is still unclear. AIM OF THE STUDY: To determine whether influx transporters contribute to the brain uptake of the highly toxic alkaloids in Aconitum including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). MATERIALS AND METHODS: The uptake of AC, MA and HA was characterized using in vitro hCMEC/D3 model and in situ mouse brain perfusion. In hCMEC/D3 cells, the effect of incubation temperature, time, initial drug concentration, energy (NaN3), extracellular and intracellular pH (FCCP and NH4Cl), the prototypical substrates/inhibitors of known organic cation transporting carriers and trans-stimulation (pre-incubating with pyrilamine and diphenhydramine) on the cellular uptake were studied. In addition, the effect of silencing OCTN1, OCTN2 and PMAT by specific siRNA was investigated. In mice, the contribution of the proton-coupled antiporter on the brain uptake of Aconitum was investigated by chemical inhibition. RESULTS: In hCMEC/D3 cells, AC, MA and HA were each taken up in a temperature-, time- and concentration-dependent manner, which were reduced by NaN3 and FCCP. Regulation of extracellular and intracellular pH as well as trans-stimulation studies showed that AC, MA and HA were transported by a proton-coupled antiporter expressed at the plasma membrane that could also transport pyrilamine and diphenhydramine. Each uptake was markedly inhibited by various cationic drugs, but insensitive to the prototypical substrates/inhibitors of identified organic cation transporting carriers, such as OCTs, PMAT, MATEs and OCTNs. In addition, silence of OCTN1, OCTN2 and PMAT had no significant inhibitory effect on the uptake of AC, MA and HA. In mice, the brain uptake of each alkaloid measured by in situ brain perfusion was suppressed by diphenhydramine when the transport capacity of P-gp/Bcrp at the BBB was chemically inhibited. CONCLUSIONS: A novel proton-coupled organic cation antiporter plays a predominant role in the blood to brain influx of AC, MA and HA at the BBB, and thus affect the safety of Aconitum species.
ETHNOPHARMACOLOGICAL RELEVANCE: The herbs of Aconitum are the essential Traditional Chinese medicine and have played an indispensable role in many Asian countries for thousands of years to treat critical illnesses, and chronic, stubborn diseases. However, Aconitum may induce severe neurotoxicity and even death. So far the mechanism of Aconitum penetrating the blood-brain barrier (BBB) is still unclear. AIM OF THE STUDY: To determine whether influx transporters contribute to the brain uptake of the highly toxic alkaloids in Aconitum including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). MATERIALS AND METHODS: The uptake of AC, MA and HA was characterized using in vitro hCMEC/D3 model and in situ mouse brain perfusion. In hCMEC/D3 cells, the effect of incubation temperature, time, initial drug concentration, energy (NaN3), extracellular and intracellular pH (FCCP and NH4Cl), the prototypical substrates/inhibitors of known organic cation transporting carriers and trans-stimulation (pre-incubating with pyrilamine and diphenhydramine) on the cellular uptake were studied. In addition, the effect of silencing OCTN1, OCTN2 and PMAT by specific siRNA was investigated. In mice, the contribution of the proton-coupled antiporter on the brain uptake of Aconitum was investigated by chemical inhibition. RESULTS: In hCMEC/D3 cells, AC, MA and HA were each taken up in a temperature-, time- and concentration-dependent manner, which were reduced by NaN3 and FCCP. Regulation of extracellular and intracellular pH as well as trans-stimulation studies showed that AC, MA and HA were transported by a proton-coupled antiporter expressed at the plasma membrane that could also transport pyrilamine and diphenhydramine. Each uptake was markedly inhibited by various cationic drugs, but insensitive to the prototypical substrates/inhibitors of identified organic cation transporting carriers, such as OCTs, PMAT, MATEs and OCTNs. In addition, silence of OCTN1, OCTN2 and PMAT had no significant inhibitory effect on the uptake of AC, MA and HA. In mice, the brain uptake of each alkaloid measured by in situ brain perfusion was suppressed by diphenhydramine when the transport capacity of P-gp/Bcrp at the BBB was chemically inhibited. CONCLUSIONS: A novel proton-coupled organic cation antiporter plays a predominant role in the blood to brain influx of AC, MA and HA at the BBB, and thus affect the safety of Aconitum species.
Authors: David A Doetsch; Salim Ansari; Ole Jensen; Lukas Gebauer; Christof Dücker; Jürgen Brockmöller; Alexandra Sachkova Journal: Int J Mol Sci Date: 2022-07-29 Impact factor: 6.208