Studies On Metabolic Characteristics Of Tanreqing Injection And Its Interaction With Sirolimus In Rats

TCM injection is a totally new dosage formation based on its long development,due to its specific characters like complicated constituents, wide therapeutic index, wehave met some serious adverse occasions. The present research background for the TCMis the quality control standards can’t match up with the current requirements, we alsodon’t have a target index to make sure the reproducibility of quality control, the basicelements of TCM hadn’t drawn great attention from the researchers. What’s more, theeffective ingredients and pharmacokinetic characters of drugs administrated in humanplasma are still unclear, we even can’t get much report about TCM’ drug-drug interactionwith other chemotherapy, all those drawbacks affect badly on the successful clinicalapplication of the TCM injection, which can’t keep up with the pace of the Modernizationof TCM, so it’s essential to improve the quality control of TCM injection and make suresafe drug administration.TRQ injection, consisting of scutellaria, pulvis ellis urs, cornu gorais, and lonicerajaponica, forsythia suspense, is targeting for lung fever syndrome and widely used astreatment for acute bronchitis, acute pneumonia in respiratory department, pediatricdepartment, surgical department, oncology department. To date there is no official qualitycontrol system for TRQ injection, we get few knowledge about its metabolism in vivo,and several repots had talked about its interaction with other chemotherapy. In this article,we took TRQ injection as the research target, via the monitor equipment such as HPLC,LC-MS/MS and LC-Q-TOF/MS, we have done some quality study of TRQ andsuccessfully determined5major components of TRQ injections, sirolimus respectively.Meanwhile, we processed further pharmacokinetic investigation of TRQ injection in vivoand its interaction with sirolimus, the detailed contents are as follow:1. We got fingerprints of11different batches of TRQ injection viaHPLC-DAD-ELSD, and finished the relative analysis, and also successfully identified53components by LC-Q-TOF/MS, and according to the specific product ion fragments, wehad distinguished some isomers, which made the fingerprints more representative. Forthe5major constituents(chlorogenic acid, caffeic acid, baicalin, ursodesoxycholic acid)of TRQ injections, a sensitive HPLC-DAD-ELSD determination method was proposed, we also validated the method, the result was satisfactory, with a calibration coefficientr2>0.99, both intra-day precision and inter-day precision below5%. We have successfullyapplied this method for determination of other TRQ injection batches. The work beforegave support for the quality control of TRQ injection and we could make sure the qualityof TRQ injection involved in the following pharmacokinetic study was stable andacceptable.2. We have identified more than20ingredients of TRQ injection admistrated intoplasma via LC-Q-TOF/MS technique. Then a LC-MS/MS method for simultaneousdetermination of5major constituents(chlorogenic acid, caffeic acid, baicalin,ursodesoxycholic acid, chenodeoxycholic acid) in mouss plasma was provided, we choseprotein precipitation as pretreatment condition, and the reagent was methanol:acetonitrile(3:1, v/v), rutin and puerarin were used as internal standard, we performed thechromatographic separation through a Zorbax SB-C18column(3.5μm,100mm×2.1mm)with gradient elution of acetonitrile and0.1%formic acid in water, the flow rate was0.3mL·min(-1), and column temperature was set at35oC. We chose MRM as the monitoringmode. The whole method was successfully validated, the calibration cure range was30(-1)4933ng·mL(-1),27(-1)3333ng·mL(-1),50-5033ng·mL(-1),550-55000ng·mL(-1),480-48000ng·mL(-1), for chlorogenic acid, caffeic acid, baicalin, ursodesoxycholic acid,chenodeoxycholic acid respectively. Both intra-day and inter-day precision were below14%, the recovery percentage ranged from70%(-1)05%. Altogether, this simple, specificand sensitive method successfully applied to pharmacokinetic study of TRQ injection,provided support for clinical therapy. Finally we primarily identified the constituents ofTRQ injection administrated via LC-Q-TOF/MS, gained some knowledge of itsmetabolism character in vivo.3. In this article, we proposed a simple, rapid, sensitive method for determinationsirolimus in mouse plasma. Protein precipitation can extract the sirolimus in whole bloodeffectively and eliminated the matrix effect. The results showed that coefficient r2wasmore than0.99of calibration curve, LOQ proved to be2.5ng·mL(-1), other parameterssuch as intra-day and inter-day precision were below15%, recovery test was alsosatisfactory. This method matched all the needs for quantitative analysis of biologicalsample, and was simple, specific, and sensitive, which can be suited for thepharmacokinetic study of TRQ injection combined with sirolimous administration. Forthe whole part of pharmacokinetic study, we had studied that the same amount of TRQ injection could affect sirolimus metabolism at different time, and the different amounts ofTRQ injection could affect sirolimus metabolism at the same time, the result suggestedthat TRQ injection promoted the Cmax, followed by the increase of AUC0-t, however itdidn’t seem to change sirolimus’s elimination in vivo.4. Via the liver microsomal incubation experiments in vitro, we had systematicallystudied whether TRQ injection and its5major constituents inhibited the activity ofCYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4and CYP2E1, thus affecting themetabolism of sirolimous in liver microsomal. The results indicated TRQ injectionaffected those6enzymes at some extent, but the5definited constituents didn’t work.Subsequently we selected liquid-liquid extraction as the pretreatment procedure, andreagents such as n-hexane, dichloromethane, acetate and water had been tested. We foundthat only water extracts inhibited the activity of enzymes, which indicated that inhibitionconstituents are strongly polar. The experiments validated that TRQ injection canimprove the bioavailability of sirolimus by inhibiting the activity of the CYP450enzymefamily, but the exact worked constituents still needed further investigation.