In vivo pharmacological efficacy of intravenously administered cepharanthine hydrochloride in rats based on its exposures
OBJECTIVE To establish a liquid chromatography massspectrometry(LC-MS/MS)method for quantitatively determining the concentration of cepharanthine in rat plasma and tissue samples after intravenous injection of cepharanthine hydrochloride.METHODS ①The LC-MS/MS method was adopted.A Phenomenex C18(3.0 mm×50 mm,2.6 μm)column was employed with a mobile phase consisting of 0.05%formic acid-2 mmol·L-1 ammonium acetate-water solution and 0.1%formic acid-acetonitrile solution under gradient elution at a flow rate of 0.6 mL·min-1.The determination was performed using positive ion multiple reaction monitoring mode assays:cepharanthine(m/z:607.3→365.1)and buspirone(IS)(m/z:386.4→122.2).② Blood samples were collected from 6 SD rats at different time points following a single iv administration of cepharanthine to determine the concentration of the drug.The main pharmacokinetic parameters were calculated using a non-compartmental model.③72 SD rats were subjected to tissue distribution experiments after a single and multiple iv administra-tion of cepharanthine,and tissue samples were collected at six different time points(n=6)for the quanti-fication of drug concentrations.④ The whole blood plasma distribution ratio(Rb/p)of cepharanthine hydrochloride(7.5 mg·kg-1)in 3 SD rats was determined 2 h after iv administration.⑤The protein binding of cepharanthine to rat plasma and lung tissue homogenates was determined by equilibrium dialysis before the concentration of the free drug within the lungs was calculated.RESULTS ① An LC-MS/MS method for quantitatively determining cepharanthine in rat plasma and tissue homogenates was devel-oped,which demonstrated an excellent linear relationship(r2>0.999)within the concentration range of 2 to 1000 μg·L-1,with a lower limit of quantification at 2 μg·L-1.The obtained results met all the require-ments for accurate quantitative detection.②The main pharmacokinetic parameters of cepharanthine in rats following a single iv administration were as follows:C0=(686.91±238.43)μg·L-1,t1/2=(29.70±6.29)h,Vz=(62.70±7.93)L·kg-1,Vss=(62.55±11.28)L·kg-1,CL=(1.50±0.23)L·h-1·kg-1 and AUC(0-t)=(4.52±0.61)h·mg·L-1.③ Concentrations in tissues exceeded those in plasma after both a single and multiple iv administration,with the highest levels in the lung.The values of AUC(0-t)in lungs were(2 547.35±156.56)and(4 481.35±479.21)h·mg·L-1 after a single and multiple iv administration,respectively.④ The content of cepharanthine in blood cells was higher than that in plasma,and Rb/p was 3.5±0.8.⑤ After correction by the protein-binding rate,the minimum concentration of free drugs in the lungs(95.04 μg·L-1)exceeded the reported antiviral activity threshold against coronaviruses(EC50=60.67 μg·L-1).CONCLUSION An LC-MS/MS method has been established to rapidly and sensitively determine the concentration of cepharanthine in rat plasma and tissues.Following intravenous administration of ceph-aranthine hydrochloride,the pulmonary exposure level of the drug is significantly higher in plasma and other tissues,providing data for evaluating its in vivo pharmacological activities.
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