布洛芬丁香酚酯前体药物的合成及其微乳给药系统的研究
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摘要
本文以减小非甾体抗炎药布洛芬的胃肠道刺激及提高中药挥发性成分丁香酚稳定性为设计思想，将药理活性具有相近或协同作用的布洛芬和丁香酚制成前体药物(布洛芬丁香酚酯，IEE)，系统地研究了该前体药物的理化性质、体内外稳定性、跨膜转运机理、在生物体内的代谢途径，在此基础上成功地制备了可供注射和口服给药的微乳制剂，对该制剂的体内药动学行为、体内分布以及药效学进行了较为深入研究，旨在探讨通过结构改造和剂型设计来提高药物疗效、降低毒副作用、改善药物稳定性等的必要性和可行性。
以布洛芬和丁香酚为原料，通过酰氯酯化反应首次合成了IEE，其化学名为丙酸，2-[4-(2-甲基丙基)苯]，2-甲氧基-4-(2-烯丙基)苯酯。该合成方法简单，回收率高，适合大批量生产。所得产物的熔点为41.2±0.5℃，结构经UV、IR、MS和~1H-NMR鉴定，确认为目标化合物。
在理化性质研究中，测定了药物在不同pH条件下的溶解度，IEE在水中的溶解度为2.98μg／ml，且受pH影响较小，属亲脂性药物，其在各种有机溶剂及油中有极好的溶解性(＞200mg／g)；油水分配系数(CLogP)为6.45；高温、高湿和光照实验结果表明，IEE稳定性良好；对药物在不同pH值溶液中的水解动力学研究显示，IEE在pH1.1-9.96范围内稳定性良好，不随pH发生改变，大于9.96时，随pH增大水解速率加快。
以Caco-2细胞模型研究了IEE的跨膜转运机理。在有酯酶抑制剂苯甲基磺酰氟(PMSF)存在的情况下IEE的P_(app)(A→B)和P_(app)(B→A)相近，分别为14.46和15.04×10~(-6)cm／s，由此初步判断IEE主要以被动扩散形式透过生物膜。分别考察了牛血清白蛋白(BSA)、PMSF、浓度、抑制剂、温度对IEE转运的影响，其中：BSA对其跨膜转运无显著性影响；在无PMSF存在的情况下，IEE在转运前和转运过程中可被体内酶水解为原形药，IEE的转运实际是由两者共同作用的结果；由于布洛芬和IEE的透过速率不同以及不同浓度条件下可供水解的酶达到饱和程度不同导致转运速率不同；P-gp抑制剂维拉帕米和MRP抑制剂吲哚美辛对IEE的转运没有显著影响，说明药物的转运不受外排泵的作用；温度对IEE的转运产生显著影响(P＜0.05)，在不同温度下由于酶的活性不同导致IEE和布洛芬的转运速率相对不同，使渗透系数发生改变。IEE的跨膜转运依赖于酶的参与，使IEE表现为较好的吸收效果。
应用LC／MS~n法首次鉴定了IEE在大鼠尿及血中的代谢产物，其中包括1种Ⅰ相代谢产物和6种Ⅱ相代谢产物。在尿中发现包括IEE和布洛芬在内的多种代谢产物，说明IEE在体内经历了多重代谢过程，静脉或口服给药1h后的血浆样品中没有发现IEE而测得布洛芬，证明IEE在生物体内的确可以水解为原形药布洛芬，但在尿样中和血样中均没有发现丁香酚原形药物的存在，推测IEE在大鼠体内主要先水解成布洛芬和丁香酚，丁香酚经进一步代谢与葡萄糖醛酸结合，LC／MS~n为IEE微乳制剂的体内药动学研究提供了科学依据。
为了促进药物吸收、提高生物利用度及减小胃肠道副作用，本文选择新型给药系统——微乳作为药物传递载体，制备了可供注射和口服的微乳制剂。分别以IPM和Miglyol 812作为油相、豆磷脂(SbPC)与HS-15为表面活性剂、PEG 400和乙醇作为助表面活性剂、重蒸水作为水相，绘制伪三元相图，研究各组分的组成对微乳形成的影响，并考察了不同油相、不同表面活性剂、助表面活性剂的组成变化对制剂粒径、粘度及稳定性的影响，确定了含有较多油相、较少表面活性剂的稳定空白微乳的组成。在空白微乳的基础上考察了药物的加入对微乳相关指标的影响，当药物含量占油相(Miglyol 812)的40％(w／w)以下时，微乳载药量可达100％，且粒径较小；当药物含量大于油相的40％以上时，微乳载药量下降并伴有药物析出，粒径变大，分布范围变宽。确定的最佳处方组成为IEE／Miglyol 812／SbPC／HS-15／PEG400／Ethanol／Water(6.4／9.6／6／6／8.4／3.6／60g)。
在体外研究的基础上，选择大鼠为实验动物，以HPLC方法为检测手段，对IEE静脉注射微乳和口服微乳进行体内药动学研究，并分别与布洛芬的两种给药途径进行了比较，以非隔室模型计算了体内药动学参数，并得出了该制剂的绝对生物利用度。IEE微乳于小鼠静注给药体内分布研究表明，肝、脾等网状内皮系统丰富器官的药物浓度明显降低，而药物在心、肺、脑的分布相对增加，但肾内药物浓度变化较小，与布洛芬水溶液组相比，平均滞留时间延长，消除相对较慢，说明该制剂具有缓释作用。
急性毒性实验结果表明，IEE微乳制剂小鼠口服和注射的LD_(50)分别为714.7和197.2mg／kg；安全性试验结果表明，该制剂不溶血、无过敏性及刺激性；药效学实验结果表明，等剂量小鼠灌胃或静注时，IEE微乳对小鼠热板法和醋酸致痛与布洛芬效果相当，对由二甲苯引起小鼠耳肿胀和角叉菜胶致大鼠足肿胀均有明显的抑制作用；小鼠胃肠道刺激性试验结果显示，灌胃给药后IEE微乳能明显降低胃肠道刺激，与布洛芬组有显著性差异(P＜0.05)，与空白组相当。
Depressing the upper gastrointestinal (GI) of NSAID and formulating poorly water-soluble drug into microemulsion delivery system were considered as two focuses in this study. Base on these two considerations, Ibuprofen eugenol ester (IEE) was synthesized. Meanwhile the physical-chemical properties, in vitro and in vivo's stability, transport mechanism and metabolism way of IEE were also investigated. IEE can be applied to mciroemulsion formulation for oral and intravenous administration. The pharmacokinetics, distribution and pharmacodynamics of the formulation were also examined in order to explore the possibility of increasing the curative effect, and decreasing the GI side effect by structure transform and formulation design.IEE [propionicacid.2-[4-(2-methylpropyl) phenyl], 2-methoxy-4-(2- propenyl) phenyl ester] was synthesized by acyl chloride method using ibuprofen and eugenol as the raw materials. The method was simple and reproducible. It can be applicable to mass production. The structure of IEE was confirmed by MS, UV, IR, ~1H-NMR spectrum, respectively. And the melting point is 41.2±0.5℃.During the physical and chemical properties studies, the solubility of the drug in different pH was determined. The solubility of IEE in water was very low (2.98μg/ml), and was not effected by pH. It belongs to poorly water-soluble lipophilic drug. However, it is easily soluble in various solvent and oil (>200mg/kg). The CLogP was 6.45. IEE shows its well stability by the results of the experiments in different temperature, humidity, and light studies. The kinetics of ester hydrolysis was studied in aqueous buffer solutions. It was stable at a wide pH range 1.10-9.96. Over this range, IEE hydrolyzed fast with pH increased.The Caco-2 cell model was utilized to examine the mechanism of IEE transport across the intestinal membrane. With the exposure to PMSF, an inhibitor of serine protease/mammalian esterase, the apparent permeability coefficients of IEE from AP-to-BL (14.46×10~(-6)cm/s) and BL-to-AP (15.04×10~(-6)cm/s) were found to be statistically indifferent (P>0.05). Therefore, IEE transported across Caco-2 cell monolayer vis an passive mechanism. The effect of BSA, PMSF, concentration, inhibitors, and temperature on the transport was investigated. It was founded that the BSA didn't affect transport of IEE in different sacs. Without the exposure to PMSF, IEE can be hydrolysis into ibuprofen before or during transport. The transport of IEE was actually due to the combine result of IEE and ibuprofen. Under various concentrations, the different P_(app) of IEE, ibuprofen and saturate degree of esterase for hydrolysis caused the differents of P_(app). The permeability coefficient was not affected when verapamil and indomethin were added, illustrating the drug was not transported via efflux mechanism. The P_(app) of IEE was affected by temperature significantly because of the different active of esterase under various temperatures. It can be concluded that the transport of IEE depends on the esterase participation. Therefore, the absorption oflEE was shown well.
IEE and its seven metabolites were first identified in rat urine and plasma with LC/MSn method, which included one phaseⅠmetabolite and six phaseⅡmetabolites. It found that the metabolites substrates included IEE and ibuprofen, which illustrated that IEE had undergone expand metabolite. No IEE but ibuprofen was found in plasma sample after 1 h iv or oral administration. It demonstrates that the IEE can be hydrolysis to ibuprofen in vivo. However, no eugenol was found both in plasma and urine sample. According to the experimental results in this study, it can be deemed that the IEE was hydrolyzed to form eugenol and ibuprofen, followed by conjugation with glucuronic acid, which forms the major metabolic pathway of IEE in rats. LC/MS~n study brought the foundation for pharmacokinetic analysis of IEE-ME.
A novel microemulsion, which administrated for oral and intravenous route, was prepared to facilitate the absorption, decrease GI side effect, and increase the bioavilability. For the mieroemulsions, Miglyol 812 was chosen as oil phase, SbPC and HS-15~(R) as surfactants (S), PEG400 and ethanol (7:3) as cosurfactant (CoS) and the double-distilled water as water phase. Pseudo-ternary phase diagrams were constructed to obtain the concentration range of each component for the microemulsion formation. The effects of various ratio of SbPC to HS-15 and different weight ratios of surfactant to cosurfactant (S/CoS) on the droplet size were investigated, as well as viscosity and stability of microemulsion. The constitute of stable blank ME with more oil and little surfactant was confirmed. The effect of drug on the ME was also examined. When the drug content was below 40％in oils (Miglyol 812), drug-loading of ME can obtained 100％with less droplet size. When the drug content was above 40％, the drug loading declined and the drug precipitation was founded. The droplet size and droplet distribution increased. The consist of optimize formulation was IEE/Miglyol 812/SbPC/HS-15/PEG 400/Ethanol/Water (6.4/9.6/6/6/8.4/3.6/60g).
The in vivo pharrnacokinetic behavior of IEE was studied utilizing HPLC method. Using the oral and intravenous route, the absolute bioavailability and pharmaeokinetic study of IEE microemulsion in rats were performed. The pharmacokinetic parameters were studied according to non-compartment model. The distribution study after intravenous administration indicated that the drug concentration in liver and spleen, RES-rich tissues after the dose of IEE-ME were significantly lower. However, the drug concentrations were higher in heart, lung and brain than those after the dose of ibuprofen solution. The prolongation of the MRT and the delay of removal of IEE-ME from circulation indicated that the formulation has sustained effect.
Acute toxicity results suggested that the LD_(50) of IEE-ME for oral and intravenous formulation were 714.7 and 197.2 mg/kg, respectively. The pharmaceutical safety test results indicated that hymolysis, simulation and pyrogen, none of these negative effects was found. The analgesic activity of IEE-ME was the same as the ibuprofen in mice utilizing hot plate method and acetic acid-induced analgesic method after the same dosage of oral and intravenous administration in pharmcodynamics test. Inhibitory effect of IEE-ME was significant by using intraplantar injection of carrageenan in rat and dimethylbenzene induced ear swelling in mice, respectively. The results of ulcerogenic activity indicated that the IEE-ME can significantly decrease(P＜0.05)the GI irritation after oral administration. It was the same as the control group.
以布洛芬和丁香酚为原料，通过酰氯酯化反应首次合成了IEE，其化学名为丙酸，2-[4-(2-甲基丙基)苯]，2-甲氧基-4-(2-烯丙基)苯酯。该合成方法简单，回收率高，适合大批量生产。所得产物的熔点为41.2±0.5℃，结构经UV、IR、MS和~1H-NMR鉴定，确认为目标化合物。
在理化性质研究中，测定了药物在不同pH条件下的溶解度，IEE在水中的溶解度为2.98μg／ml，且受pH影响较小，属亲脂性药物，其在各种有机溶剂及油中有极好的溶解性(＞200mg／g)；油水分配系数(CLogP)为6.45；高温、高湿和光照实验结果表明，IEE稳定性良好；对药物在不同pH值溶液中的水解动力学研究显示，IEE在pH1.1-9.96范围内稳定性良好，不随pH发生改变，大于9.96时，随pH增大水解速率加快。
以Caco-2细胞模型研究了IEE的跨膜转运机理。在有酯酶抑制剂苯甲基磺酰氟(PMSF)存在的情况下IEE的P_(app)(A→B)和P_(app)(B→A)相近，分别为14.46和15.04×10~(-6)cm／s，由此初步判断IEE主要以被动扩散形式透过生物膜。分别考察了牛血清白蛋白(BSA)、PMSF、浓度、抑制剂、温度对IEE转运的影响，其中：BSA对其跨膜转运无显著性影响；在无PMSF存在的情况下，IEE在转运前和转运过程中可被体内酶水解为原形药，IEE的转运实际是由两者共同作用的结果；由于布洛芬和IEE的透过速率不同以及不同浓度条件下可供水解的酶达到饱和程度不同导致转运速率不同；P-gp抑制剂维拉帕米和MRP抑制剂吲哚美辛对IEE的转运没有显著影响，说明药物的转运不受外排泵的作用；温度对IEE的转运产生显著影响(P＜0.05)，在不同温度下由于酶的活性不同导致IEE和布洛芬的转运速率相对不同，使渗透系数发生改变。IEE的跨膜转运依赖于酶的参与，使IEE表现为较好的吸收效果。
应用LC／MS~n法首次鉴定了IEE在大鼠尿及血中的代谢产物，其中包括1种Ⅰ相代谢产物和6种Ⅱ相代谢产物。在尿中发现包括IEE和布洛芬在内的多种代谢产物，说明IEE在体内经历了多重代谢过程，静脉或口服给药1h后的血浆样品中没有发现IEE而测得布洛芬，证明IEE在生物体内的确可以水解为原形药布洛芬，但在尿样中和血样中均没有发现丁香酚原形药物的存在，推测IEE在大鼠体内主要先水解成布洛芬和丁香酚，丁香酚经进一步代谢与葡萄糖醛酸结合，LC／MS~n为IEE微乳制剂的体内药动学研究提供了科学依据。
为了促进药物吸收、提高生物利用度及减小胃肠道副作用，本文选择新型给药系统——微乳作为药物传递载体，制备了可供注射和口服的微乳制剂。分别以IPM和Miglyol 812作为油相、豆磷脂(SbPC)与HS-15为表面活性剂、PEG 400和乙醇作为助表面活性剂、重蒸水作为水相，绘制伪三元相图，研究各组分的组成对微乳形成的影响，并考察了不同油相、不同表面活性剂、助表面活性剂的组成变化对制剂粒径、粘度及稳定性的影响，确定了含有较多油相、较少表面活性剂的稳定空白微乳的组成。在空白微乳的基础上考察了药物的加入对微乳相关指标的影响，当药物含量占油相(Miglyol 812)的40％(w／w)以下时，微乳载药量可达100％，且粒径较小；当药物含量大于油相的40％以上时，微乳载药量下降并伴有药物析出，粒径变大，分布范围变宽。确定的最佳处方组成为IEE／Miglyol 812／SbPC／HS-15／PEG400／Ethanol／Water(6.4／9.6／6／6／8.4／3.6／60g)。
在体外研究的基础上，选择大鼠为实验动物，以HPLC方法为检测手段，对IEE静脉注射微乳和口服微乳进行体内药动学研究，并分别与布洛芬的两种给药途径进行了比较，以非隔室模型计算了体内药动学参数，并得出了该制剂的绝对生物利用度。IEE微乳于小鼠静注给药体内分布研究表明，肝、脾等网状内皮系统丰富器官的药物浓度明显降低，而药物在心、肺、脑的分布相对增加，但肾内药物浓度变化较小，与布洛芬水溶液组相比，平均滞留时间延长，消除相对较慢，说明该制剂具有缓释作用。
急性毒性实验结果表明，IEE微乳制剂小鼠口服和注射的LD_(50)分别为714.7和197.2mg／kg；安全性试验结果表明，该制剂不溶血、无过敏性及刺激性；药效学实验结果表明，等剂量小鼠灌胃或静注时，IEE微乳对小鼠热板法和醋酸致痛与布洛芬效果相当，对由二甲苯引起小鼠耳肿胀和角叉菜胶致大鼠足肿胀均有明显的抑制作用；小鼠胃肠道刺激性试验结果显示，灌胃给药后IEE微乳能明显降低胃肠道刺激，与布洛芬组有显著性差异(P＜0.05)，与空白组相当。
Depressing the upper gastrointestinal (GI) of NSAID and formulating poorly water-soluble drug into microemulsion delivery system were considered as two focuses in this study. Base on these two considerations, Ibuprofen eugenol ester (IEE) was synthesized. Meanwhile the physical-chemical properties, in vitro and in vivo's stability, transport mechanism and metabolism way of IEE were also investigated. IEE can be applied to mciroemulsion formulation for oral and intravenous administration. The pharmacokinetics, distribution and pharmacodynamics of the formulation were also examined in order to explore the possibility of increasing the curative effect, and decreasing the GI side effect by structure transform and formulation design.IEE [propionicacid.2-[4-(2-methylpropyl) phenyl], 2-methoxy-4-(2- propenyl) phenyl ester] was synthesized by acyl chloride method using ibuprofen and eugenol as the raw materials. The method was simple and reproducible. It can be applicable to mass production. The structure of IEE was confirmed by MS, UV, IR, ~1H-NMR spectrum, respectively. And the melting point is 41.2±0.5℃.During the physical and chemical properties studies, the solubility of the drug in different pH was determined. The solubility of IEE in water was very low (2.98μg/ml), and was not effected by pH. It belongs to poorly water-soluble lipophilic drug. However, it is easily soluble in various solvent and oil (>200mg/kg). The CLogP was 6.45. IEE shows its well stability by the results of the experiments in different temperature, humidity, and light studies. The kinetics of ester hydrolysis was studied in aqueous buffer solutions. It was stable at a wide pH range 1.10-9.96. Over this range, IEE hydrolyzed fast with pH increased.The Caco-2 cell model was utilized to examine the mechanism of IEE transport across the intestinal membrane. With the exposure to PMSF, an inhibitor of serine protease/mammalian esterase, the apparent permeability coefficients of IEE from AP-to-BL (14.46×10~(-6)cm/s) and BL-to-AP (15.04×10~(-6)cm/s) were found to be statistically indifferent (P>0.05). Therefore, IEE transported across Caco-2 cell monolayer vis an passive mechanism. The effect of BSA, PMSF, concentration, inhibitors, and temperature on the transport was investigated. It was founded that the BSA didn't affect transport of IEE in different sacs. Without the exposure to PMSF, IEE can be hydrolysis into ibuprofen before or during transport. The transport of IEE was actually due to the combine result of IEE and ibuprofen. Under various concentrations, the different P_(app) of IEE, ibuprofen and saturate degree of esterase for hydrolysis caused the differents of P_(app). The permeability coefficient was not affected when verapamil and indomethin were added, illustrating the drug was not transported via efflux mechanism. The P_(app) of IEE was affected by temperature significantly because of the different active of esterase under various temperatures. It can be concluded that the transport of IEE depends on the esterase participation. Therefore, the absorption oflEE was shown well.
IEE and its seven metabolites were first identified in rat urine and plasma with LC/MSn method, which included one phaseⅠmetabolite and six phaseⅡmetabolites. It found that the metabolites substrates included IEE and ibuprofen, which illustrated that IEE had undergone expand metabolite. No IEE but ibuprofen was found in plasma sample after 1 h iv or oral administration. It demonstrates that the IEE can be hydrolysis to ibuprofen in vivo. However, no eugenol was found both in plasma and urine sample. According to the experimental results in this study, it can be deemed that the IEE was hydrolyzed to form eugenol and ibuprofen, followed by conjugation with glucuronic acid, which forms the major metabolic pathway of IEE in rats. LC/MS~n study brought the foundation for pharmacokinetic analysis of IEE-ME.
A novel microemulsion, which administrated for oral and intravenous route, was prepared to facilitate the absorption, decrease GI side effect, and increase the bioavilability. For the mieroemulsions, Miglyol 812 was chosen as oil phase, SbPC and HS-15~(R) as surfactants (S), PEG400 and ethanol (7:3) as cosurfactant (CoS) and the double-distilled water as water phase. Pseudo-ternary phase diagrams were constructed to obtain the concentration range of each component for the microemulsion formation. The effects of various ratio of SbPC to HS-15 and different weight ratios of surfactant to cosurfactant (S/CoS) on the droplet size were investigated, as well as viscosity and stability of microemulsion. The constitute of stable blank ME with more oil and little surfactant was confirmed. The effect of drug on the ME was also examined. When the drug content was below 40％in oils (Miglyol 812), drug-loading of ME can obtained 100％with less droplet size. When the drug content was above 40％, the drug loading declined and the drug precipitation was founded. The droplet size and droplet distribution increased. The consist of optimize formulation was IEE/Miglyol 812/SbPC/HS-15/PEG 400/Ethanol/Water (6.4/9.6/6/6/8.4/3.6/60g).
The in vivo pharrnacokinetic behavior of IEE was studied utilizing HPLC method. Using the oral and intravenous route, the absolute bioavailability and pharmaeokinetic study of IEE microemulsion in rats were performed. The pharmacokinetic parameters were studied according to non-compartment model. The distribution study after intravenous administration indicated that the drug concentration in liver and spleen, RES-rich tissues after the dose of IEE-ME were significantly lower. However, the drug concentrations were higher in heart, lung and brain than those after the dose of ibuprofen solution. The prolongation of the MRT and the delay of removal of IEE-ME from circulation indicated that the formulation has sustained effect.
Acute toxicity results suggested that the LD_(50) of IEE-ME for oral and intravenous formulation were 714.7 and 197.2 mg/kg, respectively. The pharmaceutical safety test results indicated that hymolysis, simulation and pyrogen, none of these negative effects was found. The analgesic activity of IEE-ME was the same as the ibuprofen in mice utilizing hot plate method and acetic acid-induced analgesic method after the same dosage of oral and intravenous administration in pharmcodynamics test. Inhibitory effect of IEE-ME was significant by using intraplantar injection of carrageenan in rat and dimethylbenzene induced ear swelling in mice, respectively. The results of ulcerogenic activity indicated that the IEE-ME can significantly decrease(P＜0.05)the GI irritation after oral administration. It was the same as the control group.
引文
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