| Entering into the body,there are two drug different effects in the BCNU[1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU,also called as carmustine)]slow-release implants.One is the biological effects the BCNU slow-release implants working on the body,including the therapeutic effects and toxic side effects drugs produced to the body,namely the pharmacodynamics and toxicology.The another is the role the drug working to the body,including drug absorption,distribution,metabolism and excretion,which is called ADME.In short,the study of pharmacokinetics about the BCNU slow-release implants,as a quantitative subject researching laws of vivo absorption,distribution,metabolism and excretion(namely vivo process)of drugs(including foreign chemicals).The study of pharmacokinetics about the BCNU slow-release implants has played an important role on the drug designed guidance,optimization of dosage regimen,dosage form improvement,and providing quick,long-lasting,low toxic and side effects of the medicine,and has revealed the laws of drug dynamic changes in the body and researching the places saving the drug and the degree of the drug savings,association ratios of plasma proteins,metabolite structure,ways of transforming metabolites and its dynamics,excretion pathway,excretion rate and etc.,providing the basis and reference for clinical rational use of drugs.Due to the rapid development of analysis technology,thus accelerating the progress of new drugs,a large number of candidate compounds are chosen into the field of drug research.Having the larger vivo toxicity,like the BCNU[1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU,also called as carmustine)]slow-release implants,or other reasons,thus causing a great waste of manpower and financial resources.In 1990's,There are about 40%compounds were eliminated,due to pharmacokinetic reasons,such as the low vivo absorption,low bio-availability,severe first pass effect,short half-life,inducing or inhibiting drug metabolism enzymes,which illustrates the important role in the pharmacokinetics in drug development.Carmustine[(2-chloroethyl(1,3-bis)-1-nitrosourea),also called as BCNU]is a nitrosourea alkylating agent,mainly used in the clinical treatment of primary and secondary brain malignant tumor,melanoma,lymphoma,malignant melanoma and lung cancer,etc.The drug has good lipid solubility,small molecular weight,being easy to penetrate the blood-brain barrier.But its effect can not play fully because of short vivo half-life.The BCNU slow-release implants is added new indications of chemical synthetic drug classification.Commissioned by the LanJin company,toxicokinetic study of the BCNU slow-release implants in liver and pancreas can be operated.Objectives:This experiment investigated the release of the BCNU slow-release implants in livers of rabbits and pancreas of rats in the drug,making clear about the drug release rate and the drug concentration of implanted site tissue and pathological changes.Methods:Establishing a simple,accurate,sensitive and specific HPLC-MS/MS method detecting the concentrations of BCNU in tissues and plasma.By using the method measuring residual concentrations of the BCNU slow-release implants in rabbit livers after implanting the implants into rabbit livers in different days,according to the 100%original concentrations in the dosage,the drug release rate is analyzed;Using the method measuring BCNU concentrations of the 8 pieces of liver tissues divided uniformly according to the degree of farness and nearness to the position implanted the implants;Using the method measuring BCNU concentrations in plasma to observe the pathological changes surrounding the liver tissues after implanting the implants and the morphological changes of rabbit liver after implanting the implants at the end of each period.By using the method measuring residual concentrations of the BCNU slow-release implants in rat pancreas after implanting the implants into rat pancreas in different days,according to the 100%original concentrations in the dosage,the drug release rate is analyzed;Using the method measuring BCNU concentrations in pancreas tissues;Using the method measuring BCNU concentrations in plasma to observe the pathological changes surrounding the pancreas tissues after implanting the implants and the morphological changes of rat pancreas after implanting the implants at the end of each period.Results:1.The confirmed methodology results for the determination of BCNU concentrations in tissues and plasma with mass spectrometryThe carmustine standard has a good linear relationship at the concentration of 2?1000 ng/mL,and the lowest limit of quantification was 2 ng/mL.The intraday precision is 2.9?6.4%,the intra-day precision is 7.3?13.4%,the accuracy is-6.4?2%.The results show that there are good intra day and inter day reproducibility in the concentration range of 2?1000ng/mL.Liver tissue samples were placed with good stability for 35 days at-20 ?,plasma samples after 3 times of freeze thawing stability.And liquid reserves placed for 35 days with good stability in-20 ?.The analysis method can meet the determination of requirements to measure the BCNU in plasma and tissue samples.2.The results of pathological changes and the release rate of the BCNU slow-release implants in rabbit liversFor the cumulative drug release concentration after implanting the implants in 7,15,30,60 days were 37.4%± 4.2%,75.2%± 12.0%,99.9%± 0.1%and 100.0%±0.0%,respectively.?the drug release is relatively slow,takes about 1 months after the release;The drug release is relatively slow,and it takes about 1 months to release it absolutely.Plasma drug concentrations were lower than the lower limit of quantification.From the results of drug concentration in liver were-tissue can show effective tumor radius of 7 days and 15 days after administration are 0.96cm±0.03cm and 1.28cm±0.11cm.The 7 days' situation after implanting medicine to liver in local exist liver sinus congestion,a large number of liver cells appeared vacuolar degeneration and cell membrane defect,and the number of inflammatory cell infiltration.Inflammatory reaction in acute phase were significantly.With the release of the drug,liver tissue,the 15 days' situation after implanting medicine to liver in local exist which normal hepatic sinusoids and lobular structure has been damaged,liver cell necrosis appeared obvious.The 30 days' scope of hepatic necrosis after implanting medicine to liver in local expand further.Pathological sections of 60 days7 situation shows that liver tissue structures of the rabbits of 1#,2#,3#,4#and 6#begin to recover normally,but the 5#,rabbits still had obvious degeneration about necrosis of liver cells,suggesting that there are some individual differences.Observing the morphological changes of the liver of rabbits after implantation of carmustine:carmustine implants expanded visible grain and had the clear outline after 7 days of implanting the BCNU slow-release implants into the rabbit livers;the clear outline was not obvious and the grains were wrapped after 15 days of implanting the BCNU slow-release implants into the rabbit livers;Two drugs became a ball,and the colloid substance were seen when the ball was broken after 30 days of implanting the BCNU slow-release implants into the rabbit livers;the drug had not seen after 60 days of implanting the BCNU slow-release implants into the rabbit livers but there still were visible white matter in liver surface,which being suspected that incomplete absorption materials were rejected to the liver surface by liver.Each time point of the liver cells were normal,suggesting that pathological changes are not caused due to surgery at each time point in the administration site of liver tissue.3 The results of BCNU(carmustine)slow-release implants about release rates and local tissue observation in rat pancreasFor the cumulative drug release concentration after implanting the implants in 7,15,30,60 days were 55.55%± 11.98%,86.43%± 7.17%,91.31%± 9.41%and 100.0%±0.0%,respectively.The drug release is relatively slow,takes about 1 month after the release;The drug release is relatively slow,and it takes about 1 or 2 months to release it absolutely.Plasma drug concentrations of rats had the trace drug distribution,and most of them were lower than the lower limit of quantification.The 7 days' situation after implanting 4mg medicine to pancreas in local exist pancreas sinus congestion,a large number of pancreas cells appeared vacuolar degeneration and cell membrane defect,and the number of inflammatory cell infiltration.Inflammatory reaction in acute phase was significantly.With the release of the drug,pancreas tissue,the 15 days'situation after implanting medicine to pancreas in local exist which normal hepatic sinusoids and lobular structure has been damaged,pancreas cell necrosis appeared obvious.The 30 days' scope of hepatic necrosis after implanting medicine to pancreas in local expand further.Pathological sections of 60 days' situation shows that pancreas tissue structures of the rabbits of 1#,2#,3#,4#and 6#begin to recover normally,but the 5#rabbits still had obvious degeneration about necrosis of pancreas cells,suggesting that there are some individual differences.Observing the morphological changes of the pancreas of rabbits after implantation of carmustine:carmustine implants expanded visible grain and had the clear outline after 7 days of implanting the BCNU slow-release implants into the rabbit pancreases;the clear outline was not obvious and the grains were wrapped after 15 days of implanting the BCNU slow-release implants into the rabbit pancreases;Two drugs became a ball,and the colloid substance were seen when the ball was broken after 30 days of implanting the BCNU slow-release implants into the rabbit pancreases;the drug had not seen after 60 days of implanting the BCNU slow-release implants into the rabbit pancreases but there still were visible white matter in pancreas surface,which being suspected that incomplete absorption materials were rejected to the pancreas surface by pancreas.The pathological observation of pancreatic administration site after 7 days,15 days,30 days and 60 days of implanting the BCNU slow-release implants into the rat pancreas found congesting pancreatic tissue,necrotic glandular tissue,inflammatory cell infiltration,obvious inflammatory reaction,vacuolar degeneration,severe immobilized cells and other acute during the reaction after 7 days of implanting the BCNU slow-release implants into the rat pancreas.Much of the necrotic pancreatic tissue lead to large area of necrosis like structures,a large number of vacuoles,and the cell structure of normal salivary gland tissue after 15 days of implanting the BCNU slow-release implants into the rat pancreas.With the release of the drug,the normal cells have not been seen without necrotic glandular tissue after 30 days of implanting the BCNU slow-release implants into the rat pancreas.The pathological observation of pancreatic administration site after 60 days of implanting the BCNU slow-release implants into the rat pancreas found that pancreatic tissue began to recover,and the necrotic cells were wrapped and devoured,and necrosis of cell were losing with creating new cells,thus seeing the normal cell structure and pancreatic tissue enters into the recovery period.Each time point of the pancreatic tissue were normal,suggesting that pathological changes are not caused due to surgery at each time point in the administration site of pancreatic tissue.Observing the morphological changes of the rat pancreas after implantation of carmustine:carmustine implants expanded visible grain and had the clear outline after 7 days of implanting the BCNU slow-release implants into the rat pancreas;the clear outline was not obvious and the grains were wrapped after 15 days of implanting the BCNU slow-release implants into the rat pancreas;Two drugs became a ball,and the colloid substance were seen when the ball was broken after 30 days of implanting the BCNU slow-release implants into the rat pancreas;the drug had not seen after 60 days of implanting the BCNU slow-release implants into the rat pancreas but there still were visible white matter in pancreas surface,which being suspected that incomplete absorption materials were rejected to the pancreas surface by rat pancreas.Measuring BCNU concentrations of the same-long pieces of liver tissues divided uniformly according to the degree of famess and nearness to the position implanted the implants cannot be carried out the smaller rat pancreatic volume,so the test determine the concentration of semi pancreatic tissue in direct.The results showed that the average drug concentration of pancreatic tissue were 8875.5±8561.7ng/g after 7 day of implanting the BCNU slow-release implants into the rat pancreas,the average drug concentration of pancreatic tissue decreased to 571.7±763.2 ng/g after 15 days of implanting the BCNU slow-release implants into the rat pancreas.Conclusion:The study establishes a simple,accurate,sensitive and specific HPLC-MS/MS method detecting the concentrations of BCNU in tissues and plasma.The carmustine standard has a good linear relationship at the concentration of 2?1000 ng/mL,and the lowest limit of quantification was 2 ng/mL.The intraday precision is 2.9?6.4%,the intra-day precision is 7.3?13.4%,the accuracy is-6.4?2%.The results show that there are good intra day and inter day reproducibility in the concentration range of 2?1000ng/mL.Liver tissue samples were placed with good stability for 35 days at-20?,plasma samples after 3 times of freeze thawing stability.And liquid reserves placed for 35 days with good stability in-20 ?.The analysis method can meet the determination of requirements to measure the BCNU in plasma and tissue samples.The results of pathological changes and the release rate of the BCNU slow-release implants shows that the cumulative drug release concentration after implanting the implants in 7,15?30,60 days were decreased with the larger tumor radius.The effective tumor radius are 0.96cm±0.03cm.The highest concentration exists in 15 days of implanting the BCNU slow-release implants.Plasma drug concentrations were lower than the lower limit of quantification and the effective tumor concentration,so there aren't systemic untoward reaction.The 7 days'and 15 days‘situations after implanting medicine to local exist pancreas sinus congestion,a large number of pancreas cells appeared vacuolar degeneration and cell membrane defect,and the number of inflammatory cell infiltration.Inflammatory reaction in acute phase was significantly.With the release of the drug,pancreas tissue,the situations after implanting medicine to local exist which normal hepatic sinusoids and lobular structure has been damaged,cell necrosis appeared obvious.The 30 days' scope of hepatic necrosis after implanting medicine to local expand furfther.Pathological sections of 60 days'situation shows that tissue structures begin to recover normally. |
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