Discovery And Evaluation Of Tanreqing Injection Quality Markers Based On Real-world Evidence

Traditional Chinese medicine（TCM）,a great treasure of the Chinese people,has age-old history of thousands of years,with both rich clinical experience and remarkable efficacy.In particular,TCM played an important role during the SARS-Co V-2 epidemic and was widely used.Although the development of TCM has ushered in new opportunities,it is more important to ensure the quality of TCM and strengthen quality control construction.Academician Changxiao Liu proposed a new concept of quality marker（Q-marker）in TCM in 2016,which pointed out a new direction for quality research.However,it is really difficult to implement the new concept,which requires a comprehensive consideration of traditional efficacy,modern pharmacology,herbal cultivation,formulae into medicine,effectiveness,and safety of Chinese medicine to find Q-markers.Most importantly,the key is to discover and confirm the Q-markers of TCM which are closely related to clinical effects,and this requires real-world research.In 2020,China food and Drug Administration issued the notice of"guiding principles for real world evidence to support drug R&D and evaluation（Trial）"（No.1 in 2020）,which clearly adopts the R&D strategy of combining real-world studies and randomized clinical trials,and provides a reference for the evaluation of human experience summaries and clinical development of TCMs for famous and old Chinese medicine practitioners’experience prescriptions and preparations for Chinese medicine medical institutions.With the rapid development of artificial intelligence technology,it has become possible to generate real-world evidence based on real-world data.Tanreqing Injection（TRQI）is composed of Scutellariae Radix（SR）,Fel Ursi（FU）,Coryu Caprae Hircus（CCH）,Forsythiae Fructus（FF）and Lonicera japonica Flos（LJF）.Since TRQI was launched in 2003,there has been a good clinical data base with over 50,000 documented case study data.Like other kinds of TCM,they are very widely used clinically and involve numerous diseases,but most of which lack sufficient clinical evidence.In particular,there were significant differences in clinical efficacy between different batches,indicating that its quality control system is not yet complete.Therefore,there is an urgent need for TRQI to conduct research on the discovery and confirmation of Q-markers associated with clinical efficacy.Therefore,this study takes TRQI as the demonstration object,adopts information technology such as artificial intelligence,and takes large sample clinical application data as the entry point to TCM and to conduct relevant evaluation studies based on Q-markers.The results are as follows:1.Discovery of Q-markers related to TRQI efficacy based on real-world evidenceFirstly,a systematic analysis of the 4,124 papers published in TRQI since it was launched until December 2019 was performed using a bibliometric approach.The results showed that 3498 papers were clinical research papers,of which 2666 were related to the treatment of respiratory diseases.In addition,the literature on the treatment of bronchitis,pneumonia,bronchopneumonia,respiratory tract infections,lung infections,and chronic obstructive pulmonary disease accounts for approximately70%of the clinical research literature and 91.64%of the literature on respiratory diseases.Therefore,TRQI is considered to be a drug primarily for the treatment of the six diseases mentioned above.Secondly,various data were collected from 898 patients suffering from the six diseases mentioned above and taking TRQI between September15,2016 and December 31,2018.Then,the XGBoost algorithm was used to model the TRQI efficacy prediction data with the aim of assessing the efficacy of different production batches of TRQI.The average correct rate of the model test set is 0.73,the average F1 score is 0.81,and the AUC value is 0.78,which proved that the model had good prediction effect.The model predicted that patients taking TRQI with production lots 1712218,1712302,1803312,and 1805204 all had significantly lower rates of improvement,while patients taking 1607114,1608308,and 1707301 all had significantly higher rates of improvement.Thirdly,a mouse model of acute lung injury was used to validate the efficacy of seven batches of TRQI with different quality levels.A comprehensive comparison of mouse lung pathology,inflammatory cell counts（leukocytes,neutrophils and mononuclear macrophages）in BALF and alterations in biochemical markers（TNF-α,IL-1β,IL-6 and SOD）in serum showed that TRQI significantly alleviated LPS-induced acute lung injury and that the efficacy of the batch with higher improvement rate was better than that of the batch with lower improvement rate,confirming the reliability of the real-world evidence-based screening results.Fourthly,the new strategy of compound analysis by UHPLC/Q-TOF-MS/MS based on multiple internal chemical libraries was used to analyze the chemical constituents of TRQI,and 126 compounds were identified.Finally,multivariate statistical analysis and metabonomics were used to screen potential Q-markers.The PCA results showed that the TRQI of batches with a high rate of improvement and batches with a low rate of improvement could be significantly distinguished.The OPLS-DA substitution test results indicated that the original model was well robust,and 15 different compounds,namely potential quality markers,were identified.Among them,adenosine,baicalin,L-pyroglutamic acid,ursodeoxycholic acid and glucose were the compounds with high improvement rate,while L-phenylalanine,L-proline,L-valine,valyl valine,L-leucine,oxylin a-7-o-gluconine,L-isoleucine,glucopyranosiduronic acid,secologanoside and chenodeoxycholic acid were the compounds with low improvement rate.2.Identification of Q-markers related to TRQI efficacy based on real-world evidenceFirstly,network pharmacology was used to screen for Q-markers.By analyzing the network topological parameters and comparing the three key parameters of Degree,Closeness Centrality and Betweenness Centrality together,the results predicted Chenodeoxycholic acid,L-Valine,Pyroglutamic acid,L-Proline and Baicalin as Q-markers.Secondly,a lipopolysaccharide-induced mouse RAW264.7 cell model was used to screen for Q-markers.The results showed that none of the administration concentrations of 40μM had any effect on macrophage survival,and Baicalin,ursodeoxycholic acid,chenodeoxycholic acid and oroxylin A-7-O-glucuronide,which predicted an inhibitory effect on nitric oxide release,were used as Q-markers.Thirdly,an in vivo model of LPS-induced acute lung injury in mice was used to screen for Q-markers.Baicalin,ursodeoxycholic acid,adenosine,and secologanoside were predicted as quality markers of efficacy by comparing the changes in mouse lung pathology,lung wet weight/dry weight ratio,and serum biochemical indices（TNF-α,IL-1β,IL-6,and SOD）.Fourthly,in vitro inhibition assays were used to compare the inhibitory effects of 15 compounds against Staphylococcus aureus and Pseudomonas aeruginosa.Lower minimum inhibitory concentrations of baicalin、leucine、pyroglutamic acid、ursodeoxycholic acid and chenodeoxycholic acid were predicted as Q-markers.Finally,the final Q-markers were determined by multidimensional comparison of radar plots.Since animal experiments are more convincing than in vitro results,a total of network pharmacology dimension,in vitro anti-inflammatory cell model dimension,in vivo mouse pathological alterations and W/D index dimension,in vivo mouse biochemical index dimension and in vitro antibacterial activity dimension were set up in the radar plot.The results showed that baicalin,ursodeoxycholic acid and chenodeoxycholic acid covered an area distinct from other compounds.The final acid and chenodeoxycholic acid.3.TRQI quality transfer studyIn this study,we proposed the research strategy of"visualization of mass transfer based on UHPLC-Q-TOF data",focusing on the quality transfer.First,the composition pattern of TRQI was investigated.The results showed that the highest proportion of TRQI was derived from SR extract,but the ionic species were not abundant,mainly baicalin;ursodeoxycholic acid and chenodeoxycholic acid were mainly derived from FU extract;amino acids and peptides were mainly derived from CCH extract;the lowest proportion was derived from LJF and FF extract,about 10%each,but the ionic species were abundant.Then,the new research strategy of quality transfer visualization was continued to investigate the transfer pattern of TRQI from"Chinese herbal pieces"to"intermediate extract"and finally to"finished formulation".The results showed that a large number of compound ions were not delivered from the Chinese herbal pieces to the extract intermediates,while the compounds were delivered from the extract intermediates to the finished formulation of TRQI with high efficiency.SR,FU,CCH,LJF and FF had 10,3,21,20 and 12 stable delivery compounds,respectively.In addition,the transfer efficiency of CCH from tablets to intermediate extract ranged from15%to 26%,with the largest fluctuations for the five herbs.4.A preliminary investigation on the resource survey and quality evaluation of the CCH The previous study showed that 7 of the 15 potential Q-markers were amino acid components,and that the conversion rate of CCH from tablets to intermediate extracts ranged from 15%to 26%,which has the largest fluctuation of the 5 herbs.However,the production process is not a problem,so I focused on the CCH.Firstly,we learned about the formation,historical research and resources of CCH by reading books and literature,communicating with goat farmers in person,and consulting with them by phone or We Chat.The results of the study showed that young goats are induced by the connective tissue and dermis above the frontal bone periosteum to form a bony keratocarpus and then a goat horn,while CCH is actually a keratin sheath formed by the epidermis wrapped around the outside of the bony keratocarpus.Historical evidence shows that CCH have a long history of being used in medicine,first seen in Shennong Ben Cao Jing,while the name of goat horns was first seen in the northern and Southern Dynasties in the Ben Cao Jing Ji Zhu.CCH resources are sufficient in China,with 69 species and60 species with goat horns,which can ensure a stable source of medicinal materials.Then,we checked the standard of CCH and showed that it was completely correct to establish the ministerial standard in 1983.However,the standard only has traits and microscopic identification,which needs to be upgraded urgently.Finally,the commercial products of CCH were examined.The results showed that the commercial herbs were mainly cashmere goats.The main components of decoction pieces are CCH shavings,CCH silk,CCH pieces and CCH powder.5.Study on the metabolic process of TRQI Q-markers in Beagle dogsIn this chapter,the metabolic processes of TRQI Q-markers in Beagle dogs will be investigated.In addition,the pharmacokinetic characterization study of Tanreqing Capsules（TRQC）,which were marketed in 2013 as an alternative dosage form of TRQI,has not been reported.Therefore,in this chapter,the metabolic profiles of baicalin,ursodeoxycholic acid and chenodeoxycholic acid in TRQI and TRQC were determined simultaneously in Beagle dogs by LC-MS/MS.A two-formulation two-cycle crossover experimental design was used to compare the daily doses of TRQC（0.09 g/kg）and TRQI（0.5 m L/kg）in six healthy Beagle dogs（half male and half female）administered as a single dose/7 consecutive days,respectively.Using puerarin as an internal standard,the blood concentration was detected using multiple reaction monitoring（MRM）mode negative ions,and data processing and statistical analysis were performed using DAS statistical software.The results showed that the three Q-markers showed good linearity in the ranges of 2.00～1000.00 ng/m L,10.00～5000.00 ng/m L and 1.00～500.00 ng/m L,with R² greater than 0.989.The AUC_{0–24 h}and AUC_t-∞values of the principal components baicalin,ursodeoxycholic acid and chenodeoxycholic acid of TRQI and TRQC were evaluated by ANOVA with double one-sided t-test.There were no significant（P>0.05）differences in AUC_{0-24 h} and AUC_t-∞between the two preparations.Therefore,the exposure of baicalin,ursodeoxycholic acid and chenodeoxycholic acid in Beagle dogs at the administered doses was consistent between TRQC and TRQI.