| Objective:Non-small cell lung cancer（NSCLC）accounts for 80～85%of lung cancers.About 50%of patients have brain metastases.Their natural survival period is as low as7 weeks and the prognosis is extremely poor.However,there is currently no good drug treatment for NSCLC brain metastasis.Gefitinib has good efficacy in the treatment of NSCLC,with fewer adverse reactions and low price.However,it is difficult to enter the brain.The blood-brain barrier permeability is about 1.1～1.4%.The efficacy of treatment of NSCLC brain metastases is limited.Therefore,it is urgent to study a new type of oral drug delivery system to improve the brain distribution.Due to the difficulty of gefitinib entering the brain,this article used the original research product IRESSA?as the reference preparation,based on the oral self-microemulsion delivery system to solubilize gefitinib,and screened and optimized the prescription.With this system as the research object,we conducted research on appearance characterization and other formulation characteristics,in vivo and in vitro pharmaceutical behavior and mechanism preliminary research,in order to obtain a new type of gefitinib oral self-microemulsion delivery with higher brain distribution than the original product IRESSA?system.Methods:1.Establishment and optimization of gefitinib oral self-microemulsion delivery system.In the solubility test of gefitinib in the emulsifier,co-emulsifier and oil phase,we selected the excipients with superior solubility.In the compatibility test of the excipients,we determined the emulsifier and oil phase types based on the emulsification time and the light transmittance after the emulsification was completed.By drawing pseudo-ternary phase diagram and using the largest self-microemulsion area as the evaluation index,we determined the type of co-emulsifier.By using response surface method,we evaluated the emulsification time,light transmittance and encapsulation rate after emulsification determine the oil phase ratio and Km value.Finally,we successfully established an oral self-microemulsion delivery system for gefitinib（GFB-SMEDDS）.2.In vitro characterization studies of gefitinib oral self-microemulsion delivery system.We observed the appearance of self-microemulsion by naked eyes and used Malvern laser particle size analyzer to determine particle size and distribution.We used differential scanning calorimetry and fourier infrared spectroscopy to characterize the inclusion state of gefitinib self-microemulsion.Through the establishment of ultraviolet analysis method,the stability and drug loading of gefitinib self-microemulsion in vitro simulated in vivo under different physiological p H were quickly investigated.3.Preliminary study in mice on the pharmaceutical behavior of gefitinib oral self-microemulsion delivery system.We established a specific,rapid and sensitive HPLC detection method to determine the content of gefitinib in biological samples,standardized and verified the specificity,linearity,recovery,repeatability and stability of the method.Our team compared the pharmacokinetic behavior and tissue distribution of the oral self-microemulsion delivery system and the reference preparation IRESSA?in KM mice,draw the drug-time curve,and clarify the brain distribution status.4.Preliminary study on the mechanism of gefitinib oral self-microemulsion delivery system in rat gastrointestinal absorption.We established a HPLC method for the determination of gefitinib in rat gastric perfusion and intestinal perfusion,and verified the specificity,linearity,recovery,precision and stability of the method.Through gastric perfusion test,intestinal perfusion test,and intestinal ectropion test in SD rats,the gastrointestinal absorption of gefitinib from microemulsion,raw materials and reference preparation IRESSA?was explored,and the oral administration absorption mechanism of gefitinib was preliminarily explored.Results:1.Solubility test and excipient compatibility test determined that the oil phase was GTCC,and RH-40/LS-610=1:1 was used as the emulsifier and the pseudo-ternary phase diagram from the largest area of the microemulsion area was used as the index to determine PEG-400 as co-emulsifier.Response surface test determined that optimal prescription was Gefitinib:RH-40:LS610:PEG-400:GTCC=1:26:26:35:12,which was named LLP610.2.The self-microemulsion sample of gefitinib was clear and transparent,with good fluidity.Its particle size was 13.40±0.06 nm,and the polydispersity coefficient（PDI）was 0.154±0.006,indicating that the particle size of the self-microemulsion sample of gefitinib was small,which could form a nanoemulsion with a narrow and uniform distribution.Differential scanning calorimetry and Fourier infrared spectroscopy showed that the gefitinib molecule was encapsulated in a microemulsion in an amorphous state.The in vitro release test results showed that in a simulated body under physiological conditions,a large amount（≥70%）of gefitinib existed in the form of microemulsion droplets,and a small amount（≤30%）existed as free molecules.Gefitinib self-microemulsion was in good condition at different physiological p H.3.Using IRESSA?as the reference preparation,the pharmacokinetic behaviorstudy in mice showed that after a single oral administration,gefitinib self-microemulsion plasma AUC（0-48h）=114.41±8.02μg.h/m L,IRESSA?AUC（0-48h）=93.89±9.01μg.h/m L,relative bioavailability was 119%.Tissue distribution studies showed that gefitinib self-microemulsion brain AUC（0-48h）=81.01±6.02μg.h/m L,IRESSA?brain AUC（0-48h）=37.89±3.14μg.h/m L,indicating that the distribution of the self-microemulsion brain was significantly higher than that of IRESSA?.SMEDDS brain t1/2=7.39±0.14 h,IRESSA?brain t1/2=2.01±0.12 h,compared with IRESSA?,SMEDDS significantly prolonged the action time of gefitinib in the brain.The concentration of gefitinib in mouse brain was higher than IRESSA?,Cmax was 1.20 times that of IRESSA?,and the targeting index of brain tissue TI was 214%,and the relative targeting efficiency RTE was 123%,both of which were the highest among all tissues.These results indicated that gefitinib self-microemulsion had a clear tendency of brain aggregation and a higher brain tissue distribution.4.The results of gastric perfusion test showed that gefitinib self-microemulsion,IRESSA?and API were all absorbed in the stomach of rats.The net absorption percentage of API and IRESSA?decreased with the concentration increased,and self-microemulsions followed with the concentration increased,the percentage of net absorption first increased and then remains stable.It means that absorption of self-microemulsion in the stomach was not simplely passive diffusion,but also included carrier media transport.The results of intestinal perfusion test showed that gefitinib self-microemulsion,IRESSA?And the drug substance was absorbed in the small intestine（duodenum,jejunum,ileum）and colon at different p H.The absorption percentages of the three systems in the duodenum were 26%,14%and 8%respectively.Both were the highest in each intestinal segment,indicating that the absorption of gefitinib was related to the physiological p H of each intestinal segment,and the p H increases and the absorption effect became worse.The absorption rate constant（Ka）of self-microemulsion in the duodenum was 2.80 times and 4.46 times that of IRESSA?and API,respectively;Apparent permeability coefficient（Pa PP）was 1.87 times and2.98 times that of IRESSA?and API respectively.The percentages（P%）were 1.91times and 3.21 times that of IRESSA?and API respectively.At the same time,the Ka,Pa PP and P%of gefitinib self-microemulsion in the remaining three intestinal segments were significantly higher than IRESSA?and API.The self-microemulsion system could effectively increased the rate and extented of drug absorption in each intestinal segment.Conclusion:In view of the difficulty in getting gefitinib into the brain and the current situation that there was no good drug treatment for NSCLC brain metastasis,we had successfully established the oral self-microemulsion delivery system of gefitinib by screening and optimizing prescription of gefitinib self-microemulsion,which has a significant advantage in brain tissue distribution compared with the original research product IRESSA?,and completed preliminary exploration of its absorption mechanism in the body.The brain AUC（0-48h）of this system was 2.14 times that of IRESSA?,and it had obvious brain distribution of advantage,laying a good foundation for the development of drugs for NSCLC brain metastasis. |
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