The Exposure Characteristics And Mechanism Of Tetrandrine Against Pulmonary Infection In Different Delivery Routes

BackgroundTetrandrine is a natural medicine extracted from the root of Stephania tetrandra,which has been used clinically for decades to treat silicosis,rheumatism,pain,and lung cancer.With the outbreak of SARS,MERS,and COVID-19 in recent years,there is a great need for drugs to treat respiratory infectious diseases caused by coronaviruses.Recent studies have shown that tetrandrine may inhibit virus entry into host cells by blocking the two-pore channel 2（TPC2）and exhibit good activity against coronaviruses such as SARS-CoV-2 in vitro(EC₉₀=7.65μM in Vero E6 cells,MOI=0.05).However,the researches on its antiviral activity are at the cellular level.The organism is like a"black box"system composed of multiple biological barriers,whether tetrandrine can exert antiviral effects in vivo is full of unknowns.However,there is currently a lack of system studies on the exposure and disposition characteristics of tetrandrine in animals and humans,and conflicting results among different literatures.Therefore,this study aims to use pharmacokinetic technology to investigate the exposure characteristics and changes of tetrandrine under different administration routes at different levels,from cells to whole animal models.Combining with the pathological and physiological conditions of lung infection-induced inflammation,the potential of tetrandrine to exert anti-viral effects against pulmonary infectious diseases will be comprehensively evaluated in vivo.Based on the physicochemical properties and in vivo exposure characteristics of tetrandrine,a physiologically based pharmacokinetic（PBPK）model will be established to predict the exposure level in humans and evaluate the potential application prospect of tetrandrine against COVID-19 and other pulmonary infectious diseases,which also provides data support for the rapid repositioning of tetrandrine against other viral infections in the future.Objectives1.Clarify the exposure and disposition characteristics of tetrandrine in animals;2.Determine whether inhalation administration can enhance the lung-targeting effect of tetrandrine;3.Explore the influence and mechanism of inflammation on the exposure and disposition characteristics of tetrandrine in vivo;4.Investigate the mechanism of high tissue distribution of tetrandrine;5.Use a PBPK model to stimulate the exposure of tetrandrine in humans;6.Evaluate the feasibility of tetrandrine as a treatment for SARS-CoV-2 using PBPK modeling.Methods1.The exposure and disposition characteristics of tetrandrine in animals was studied.（1）A LC-MS/MS method was established and validated to measure the concentration of tetrandrine in plasma samples.Pharmacokinetic experiments were conducted in rats and beagles via intravenous and oral administration to determine basic pharmacokinetic parameters.（2）The distribution of tetrandrine in different tissues after oral and inhalation administration was compared,aiming to clarify the tissue distribution characteristics dependent on the administration route.Tissue concentration in rats after intragastric administration and inhalation was determined to clarify its route-dependent biodistribution characteristics.（3）Various in vitro incubation models using different species of liver microsomes and recombinant metabolic enzymes were employed to investigate the metabolic stability,metabolic enzyme phenotype,and inhibitory effect of tetrandrine on metabolic enzymes.（4）Metabolic cages were used to collect feces,urine,and bile samples to study the excretion and material balance of tetrandrine after oral administration in rats.2.The mechanism of high tissue distribution of tetrandrine was explored.（1）The lysosomal inhibitor monensin was used to study the effect of lysosome trapping on tetrandrine in A549,Hep G2,RAW264.7 and C8-D1A cells.（2）HEK293 cells overexpressing uptake transporters OATP1B1 and 1B3 were employed to study the uptake of OATP transporters on tetrandrine.（3）Caco-2 and LLC-PK1 monolayer cell models were built with the help of Transwell device to investigate the bidirectional membrane permeability of tetrandrine.（4）LLC-PK1 cells overexpressing the efflux transporter MDR1 were used to study the efflux effect of the MDR1 transporter on tetrandrine.3.The influence of inflammation on the disposition of tetrandrine in vivo was investigated.（1）The acute pneumonia mouse model induced by inhalation of lipopolysaccharide was established to study the effect of inflammation on the concentration of tetrandrine in blood and tissues.（2）Liver and lung microsomes were separated from normal and acute pneumonia mice by differential centrifugation method to study the influence of inflammation on metabolic enzymes.（3）Inflammation cell models were established using cells from different tissues to study the effect of inflammation on cellular uptake of tetrandrine and lysosomal trapping.4.A PBPK model was established using the PBPK software Gastro Plus to simulate the exposure of the tetrandrine in human,based on its physical and chemical properties and the pharmacokinetic data of tetrandrine in animals.The model predicts the anti-viral prospects of tetrandrine in vivo by combining the anti-SARS-CoV-2 pharmacodynamic data in vitro with the effective exposure in lungs.Results1.The exposure and disposition characteristics of tetrandrine in animals were obtained.（1）The plasma concentration of tetrandrine in rats and beagle dogs is relatively low,but the apparent volume of distribution is extremely high.（2）Tetrandrine is mainly distributed in tissues rather than blood circulation in rats,and the concentrations in liver,spleen,lung,kidney,and intestine are much higher than in other tissues,and inhalation administration can increase the exposure to the lungs and reduce exposure to other tissues.（3）Liver clearance rates in rats and dogs were relatively low calculated by intrinsic clearance rates acquired in liver microsomes from various species,which was consistent with the in vivo results.Tetrandrine is mainly metabolized by CYP3A,and can inhibit some metabolic enzymes in tissues,which may be a risk of drug-drug interaction.（4）Tetrandrine is eliminated through metabolism in vivo,and excreted mainly in the form of metabolites rather than prototype.2.It was verified that the bio-distribution characteristics of tetrandrine are mainly related to the lysosomal trapping and MDR1 efflux transporter.（1）After adding the lysosomal inhibitor monensin,the uptake of tetrandrine in cells from various tissues was significantly inhibited,suggesting that lysosomal trapping is one of the main pathways for tetrandrine to enter cells.（2）The uptake of tetrandrine in HEK293 cells over expressing OATP1B1 and 1B3 had no difference with wild-type HEK293 cells.（3）The efflux rate of tetrandrine in LLC-PK1 cells over expressing MDR1 was much greater than wild type,and it could be significantly inhibited by MDR1 inhibitors,indicating that tetrandrine is the substrate of MDR1.3.Inflammation can inhibit the metabolism of tetrandrine,thereby increasing the exposure in vivo.（1）The acute pneumonia mouse model induced by inhalation of lipopolysaccharide was successfully established.The exposure of tetrandrine in vivo,especially in lungs,was significantly increased,and the clearance rate was significantly decreased;（2）The activity of CYP3A in liver microsomes extracted from acute pneumonia mice was much lower than normal level,and the turnover rate of tetrandrine in liver microsomes of mice with acute pneumonia was markedly reduced.（3）The uptake of tetrandrine in A549 cells（derived from lung tissue）increased in the inflammatory state,but inflammation hardly affected the intensity of lysosomal trapping.4.The PBPK model was used to predict the effective treatment dosage and route of administration for tetrandrine in the real world.By bridging the in vitro anti-SARS-CoV-2 data and effective drug concentration data in rats’lungs,inhalation administration was predicted to achieve an anti-COVID-19 effect in rats’lungs.In addition,a PBPK model for oral administration was successfully established,and it was predicted that the lung’s free drug concentration could reach an effective concentration of anti-SARS-CoV-2 after taking 100mg,three times a day for six consecutive days,according to the current drug instructions for the treatment of silicosis.ConclusionThis study systematically investigated the exposure and disposition characteristics of tetrandrine in animals.It was found that tetrandrine had a wide distribution in various tissues due to lysosomal capture and MDR1 efflux,especially in tissues with abundant lysosomes such as liver,spleen,lung,kidney,and intestine,but with low concentrations in the brain.The distribution of tetrandrine in animals was closely related to the administration route,and inhalation administration could enhance its lung-targeting effect.The free drug concentration in the lung could reach the level of anti-SARS-CoV-2 in vitro,which was expected to play a role in the treatment of COVID-19.In an inflammatory state,the exposure of tetrandrine in the body,especially in the lungs,was significantly increased due to the inhibition of its metabolism,which may be more conducive to its therapeutic effect in the lungs.By establishing a physiologically based pharmacokinetic（PBPK）model for oral administration of tetrandrine in humans,this study provided the first conversion of drug exposure from animals to humans,which provided an important reference for predicting the feasibility of anti-SARS-CoV-2 in humans and selecting dosages for future clinical use.It also provided ideas for preclinical studies of similar drugs.