The Pharmacokinetic Study Of Triblock Copolymer PLGA-PEG-PLGA

With the rapid development of drug delivery system（DDS）,pharmaceutical polymer excipients have been widely developed and applied in the field of pharmaceutical preparations.Pharmaceutical polymer excipients have a series of advantages,such as improving solubility,enhancing targeting,reducing toxicity and improving the pharmacokinetic behavior of supported drugs.However,more and more studies have found that some pharmaceutical polymer excipients in recent years,which were generally considered to be bioinert can interact with the body to produce activity or toxic side effects.Therefore,both the National Drug Administration of China and the U.S.Food and Drug Administration require a comprehensive pharmacokinetic evaluation of pharmaceutical polymer excipients,which is of great significance for evaluating their in vivo biosafety and improving the clinical conversion success rate of DDS.Poly（lactide-co-glycolide）-block-poly（ethyleneglycol）-block-poly（lactide-co-glycolide）（PLGA-PEG-PLGA）is a kind of triblock pharmaceutical polymer excipients with good biocompatibility and biodegradability,which can produce thermosensitive gelation effect under certain composition and proportion conditions.Therefore,it has broad development and application prospects in the field of drug delivery.At present,the research on PLGA-PEG-PLGA hydrogel DDS mainly focuses on prescription design,biological effects and pharmacokinetic evaluation.Because PLGA-PEG-PLGA is a complex triblock copolymer with high molecular weight and mass polydispersion,the development of full-profile quantitative and qualitative analysis methods is a great challenge.Therefore,the comprehensive pharmacokinetic evaluation of PLGA-PEG-PLGA is still blank.It greatly restricts the design and development of DDS based on PLGA-PEG-PLGA polymer excipients.To solve the above problems,liquid chromatography-tandem mass spectrometry（LC-MS/MS）was used in this study.By increasing the declustering potential（DP）in the ion source,dimensionality reduction treatment of various polymer precursor ions was processed and a new method for the quantitative analysis of the full profile components of PLGA-PEG-PLGA triblock complex copolymers by multilpe reaction monitoring（MRM）scanning was successfully established.Based on this method,the comparative pharmacokinetics of thermosensitive hydrogel DDS with PLGA-PEG-PLGA and PLGA-PEG-PLGA-loaded irinotecan（IRN）were studied in rats.This study found that there is an interaction between PLGA-PEG-PLGA excipients and loaded drugs for the first time.In other words,excipients can affect the plasma pharmacokinetic behavior of loaded drugs,and at the same time,loaded drugs can also affect the plasma pharmacokinetic behavior of excipients.The main research contents and results of this study are as follows:（1）Study on the mass spectrometry fragment regularity of PLGA-PEG-PLGA and PEG 1500The mass distribution of PLGA-PEG-PLGA and its metabolite PEG 1500 was studied by high resolution time of flight mass spectrometry.By adjusting the DP in the ion source and the collision energy（CE）in Q2,the PLGA-PEG-PLGA and PEG 1500precursor ions did not fragment and was detected in intact form.Then the mass distribution information of PLGA-PEG-PLGA and PEG 1500 was obtained.The results showed that due to the structural characteristics of PLGA-PEG-PLGA triblock polymer and the complex types and quantities of its adduct ions,only a fuzzy normal mass distribution can be observed.The mass distribution center is about 5200 Da,which accords with the actual molecular weight of PLGA-PEG-PLGA.In addition,the main in vivo metabolite of PLGA-PEG-PLGA,PEG 1500 mainly exists in the form of carrying double and triple charges.The mass normal distribution of the double and triple charged ions respectively can be observed,and the distribution center is about1500 Da,which accords with the actual molecular weight of PEG 1500.The fragment pattern of PLGA-PEG-PLGA and PEG 1500 in the ion source and in Q2 was also investigated by adjusting the values of DP and CE.The results showed with the increase of DP,PLGA-PEG-PLGA gradually produces different series of fragment ions,including[LA_n+H]⁺,[GA+LA_n+H]⁺,[EG_n+H]⁺and other fragment ions which may have multiple affiliations.With the increase of CE in Q2,the above fragment ions were also obtained.For PEG 1500,PEG characteristic fragment ions at m/z89.0567,133.0828,177.1098,221.1362,265.1612,309.1880 and 353.2149 could be observed with the increase of DP in the ion source.With the increase of CE in Q2,the above-mentioned PEG characteristic fragment ions could also be observed.The study of the fragment regularity of PLGA-PEG-PLGA and PEG 1500 by mass spectrometry provides a good technical foundation for the subsequent establishment of the full profile quantitative LC-MS/MS analysis method of biological samples based on in-source CID-MRM.（2）Plasma pharmacokinetics of PLGA-PEG-PLGA and IRN in ratsA novel LC-MS/MS method for simultaneous quantification of PLGA-PEG-PLGA and its metabolite PEG 1500 in different biological substrates was established by in source-CID-MRM strategy.In addition,a new LC-MS/MS method for simultaneous quantification of IRN and its active metabolite 7-ethyl-10-hydroxycamptothecin（SN38）in rat plasma was established.The established analytical methods has good selectivity and reproducibility,and has passed the methodological verification.The methods are suitable for further in vivo pharmacokinetics studies of PLGA-PEG-PLGA and IRN.Using the established analytical methods above,the pharmacokinetics of rats in this study found that:after subcutaneous injection of 20 wt%PLGA-PEG-PLGA of200 mg/kg,the concentration of PLGA-PEG-PLGA was lower than the lower limit of quantitation（LLOQ）at all time points.On the contrary,the main metabolite PEG 1500was higher than LLOQ,which t_1/2 could reach 68.8 h.These results suggest that after subcutaneous injection of PLGA-PEG-PLGA,the in-situ gel formed at the injection site can maintain a long release time.PLGA-PEG-PLGA is rapidly metabolized in vivo.And main metabolite PEG 1500 is exposed at the systemic level,rather than PLGA-PEG-PLGA.After subcutaneous injection of 20 wt%PLGA-PEG-PLGA+IRN of 200 mg/kg（IRN concentration of 5 mg/kg）,no significant difference was observed in AUC(_0-t)and t_1/2 of PEG 1500 compared with subcutaneous injection with equal dose of blank PLGA-PEG-PLGA（P>0.05）.However,both C_max and T_max decreased more than two times,indicating that drug loading affected the pharmacokinetic behavior of PLGA-PEG-PLGA.This result is a new discovery in the study of the interaction between polymer excipients and loaded drugs,which is of great significance to enrich and develop the connotation and knowledge boundary of pharmacokinetic research.In addition,this finding also strongly suggests that when conducting pharmacokinetic studies on pharmaceutical polymer excipients,pharmacokinetic studies should be conducted on blank excipients that are not loaded with drugs,and pharmacokinetic studies should also be conducted on pharmaceutical excipients in drug-loaded preparations at the same time to determine whether there is pharmacokinetic interaction between drugs and excipients.After intravenous injection and oral administration of 5 mg/kg IRN solution in rats,it was observed that the bioavailability of IRN in the oral group was very low,only about 2.3%.But the IRN bioavailability in the subcutaneous PLGA-PEG-PLGA+IRN group was close to 90%.The AUC(_0-t)of SN38 even exceeded that of SN38 after intravenous injection of IRN solution.In addition,the C_max of IRN and SN38 after subcutaneous injection of PLGA-PEG-PLGA+IRN was lower than that in the intravenous injection group,and the t_1/2 of IRN was extended from 3 h to 12 h.This suggests that hydrogel can reduce the IRN administration frequency and significantly reduce the risk of acute toxicity of IRN,which is expected to improve patient compliance if used clinically in the future.（3）Study on tissue distribution of PLGA-PEG-PLGA in ratsA new quantitative method for PLGA-PEG-PLGA and its main metabolite PEG1500 in rat tissues was established and validated.The results of tissue distribution experiment showed that after subcutaneous injection of 20 wt%PLGA-PEG-PLGA of200 mg/kg,the concentration of PLGA-PEG-PLGA in all tissues and organs was lower than LLOQ,and its main metabolite PEG 1500 was mainly distributed in the kidney.and less distributed in other tissues,and only a low concentration of PEG 1500 was detected in some samples at 0.5 h and 1.5 h.At 0.5 h,the concentration of PEG 1500in each organ was ordered as follows（ng/g）:kidney（2746）>stomach（460.5）>liver（273.5）>heart（130.0）.At 1.5 h,the concentration of PEG 1500 in each organ was ordered as follows（ng/g）:kidney（3332）>stomach（391.5）>lungs（227.5）>liver（131.5）>fat（122.5）.At 48 h,the concentration of PEG 1500 in all tissue samples was lower than LLOQ.Because of the high concentration of PLGA-PEG-PLGA in kidney over a short time,the effects of PLGA-PEG-PLGA on renal insufficiency patient and the potential of acute renal toxicity should be concerned.（4）Study on metabolism and excretion of PLGA-PEG-PLGA in ratsThe metabolic pathway of PLGA-PEG-PLGA in rats was investigated by Q-Q-TOF MS,and a quantitative method for the determination of PLGA-PEG-PLGA and PEG 1500 in urine and fecal samples of rats was established.After subcutaneous injection of 20 wt%PLGA-PEG-PLGA of 200 mg/kg,the main metabolite in rats was PEG 1500.PLGA-PEG-PLGA is mainly excreted in urine in the form of PEG 1500.After 14 days,the cumulative excretion curve of urine was close to the plateau,and the cumulative excretion rate was 44.80%,of which 44.78%was PEG 1500.Only less than0.02%was the original form of PLGA-PEG-PLGA.The PEG 1500 excreted through feces reached the plateau at 3 days,and the cumulative excretion rate was 2.60%.The reason for these results is that after subcutaneous injection of PLGA-PEG-PLGA,thermosensitive hydrogel can be formed quickly,and the absorption rate is slow,resulting in less PLGA-PEG-PLGA entering the rat system circulation at the same time.The PLGA blocks at both ends of PLGA-PEG-PLGA can be first metabolized to lactic acid and glycolic acid by esterase,further metabolized to CO₂ and H₂O,and then quickly excreted from the body.The PEG 1500 blocks produced by PLGA-PEG-PLGA metabolism are relatively stable in the body and are mainly excreted through the kidney in their original form.（5）Study on the inhibitory effect of PLGA-PEG-PLGA and PEG 1500 on CYP450enzymeThe quantitative analysis method for 8 characteristic substrates metabolites corresponding to CYP3A4,CYP2C8,CYP2C9,CYP2D6,CYP2C19,CYP2B6 and CYP1A2 of CYP450 enzyme and the in vitro incubation system of human liver microsomes were established.The inhibitory effects of PLGA-PEG-PLGA and its metabolite PEG 1500 on 7 CYP450 enzymes were studied by mixed probe substrate method.The results showed that PLGA-PEG-PLGA had no significant inhibition on CYP1A2,CYP2B6,CYP2C9,CYP2C19 and CYP2D6.It has weak inhibitory effect on CYP2C8 and CYP3A4.PEG 1500 has no significant inhibition on CYP2B6 and CYP2C8.It has weak inhibitory effect on CYP1A2,CYP2C9,CYP2C19,CYP2D6 and CYP3A4.Considering that PLGA-PEG-PLGA is a medicinal excipient,which may be needed of a large dosage in clinical applications,it still needs to be vigilant of the possibility of excipient-drug interactions based on drug metabolizing enzymes.In summary,this study innovatively developed a new LC-MS/MS quantitative method for the full profile components of PLGA-PEG-PLGA in biological samples,systematically studied its pharmacokinetic behavior in rats,and conducted a comparative study on the pharmacokinetic behavior of drugs and excipients.The triblock polymer analysis method established in this study can provide a useful research strategy for the development of polymer pharmaceutical excipients with complex structure,large molecular weight and polydispersion.The research strategy and results obtained in this study will help to enrich and develop the basic theory and methods of pharmacokinetics.