拓?fù)涮婵捣聪嘀|(zhì)納米�？诜苿┭芯�
發(fā)布時間：2018-08-27 05:47
【摘要】：拓?fù)涮婵?TPT)是水溶性半合成喜樹堿(CPT)類抗癌藥物,可以特異性抑制拓?fù)洚悩?gòu)酶Ⅰ的功能,阻礙DNA的復(fù)制。目前拓?fù)涮婵瞪鲜袆┬陀型負(fù)涮婵底⑸溆脙龈煞坩槃┖涂诜z囊劑。相比靜脈注射給藥而言,口服TPT臨床治療有效、方便,且具有較低的骨髓抑制作用,但由于拓?fù)涮婵祷钚詢?nèi)酯結(jié)構(gòu)受pH影響,在中性及堿性條件下易水解開環(huán)為羧酸鹽構(gòu)型,從而失去活性,導(dǎo)致生物利用度降低,抗腫瘤活性下降,所以選擇合適的藥物載體在腸道中對其活性構(gòu)型進(jìn)行保護(hù)顯得尤為重要,另一方面,拓?fù)涮婵悼诜缶哂休^強的胃腸道不良反應(yīng)。本課題針對現(xiàn)有TPT口服制劑存在的問題,研究制備拓?fù)涮婵捣聪嘀|(zhì)納米粒新劑型,目的是增加藥物在消化道中的穩(wěn)定性同時減少胃腸粘膜刺激,并且提高藥物的口服生物利用度。通過單因素試驗與正交試驗,以透光率、粒徑、澄清度等為指標(biāo),對反相脂質(zhì)納米粒的膜材種類、用量以及載藥量進(jìn)行了篩選,采用冷凍干燥工藝、以中鏈甘油三酸酯(MCT)為油相制備反相脂質(zhì)納米粒(RLBN-TPT)油溶液,載藥量為4mg/g。通過動態(tài)光散射和透射電鏡對RLBN-TPT制劑進(jìn)行了表征,結(jié)果顯示反相脂質(zhì)納米粒的粒徑為60nm左右,呈均勻球狀。RLBN-TPT制劑在模擬腸液中分散后,對其粒徑和微觀形態(tài)進(jìn)行了觀察,結(jié)果顯示分散后形成均勻球形的脂質(zhì)囊泡或乳滴。在體外穩(wěn)定性評價研究中,建立了高效液相色譜法同時測定拓?fù)涮婵甸_環(huán)、閉環(huán)兩種構(gòu)型的含量。通過測定拓?fù)涮婵捣聪嘀|(zhì)納米粒(RLBN-TPT)在模擬腸液中的釋放,研究拓?fù)涮婵翟谌斯つc液中的穩(wěn)定性,考察反相脂質(zhì)納米粒對藥物的保護(hù)作用。選用CAPCELL PAK C18 MGⅡ色譜柱(250×4.6 mm,5μm),流動相為A:0.1%三乙胺(用冰醋酸調(diào)節(jié)pH值至6.4)-B:乙腈[梯度:0~8min(88%A~60%A);8~17(60%A)],流速1.0mL/min,檢測波長360nm,柱溫55℃。結(jié)果表明開環(huán)、閉環(huán)構(gòu)型濃度在0.25~5.00μg·mL-1范圍內(nèi),濃度與峰面積呈良好線性關(guān)系,該方法回收率高,準(zhǔn)確度好。與拓?fù)涮婵邓芤罕容^,反相脂質(zhì)納米粒中的拓?fù)涮婵翟谀c液中的穩(wěn)定性明顯增強,開環(huán)構(gòu)型產(chǎn)生的速率明顯減小(p0.05),表明反相脂質(zhì)納米粒對藥物活性內(nèi)酯形式具有一定保護(hù)作用,有效避免其在短時間內(nèi)的水解開環(huán),增強拓?fù)涮婵翟谀M腸液中的穩(wěn)定性。建立了HPLC法測定RLBN-TPT制劑中拓?fù)涮婵档暮?方法學(xué)研究表明,拓?fù)涮婵捣迕娣e和濃度在0.125μmg·mL-1~125μg·mL-1范圍內(nèi)呈良好的線性關(guān)系,且高、中、低的回收率均在98%-102%之間,RSD為0.67%,該方法準(zhǔn)確、精密度高、專屬性強、靈敏度高,可以用于制劑中拓?fù)涮婵岛康臏y定;又建立了HPLC法測定RLBN-TPT制劑中拓?fù)涮婵档挠嘘P(guān)物質(zhì),方法學(xué)研究表明該方法靈敏度高、專屬性強、精密度高,可以用于制劑中拓?fù)涮婵涤嘘P(guān)物質(zhì)的測定。通過影響因素試驗結(jié)果可知,RLBN-TPT制劑對濕、熱比較敏感,所以在儲存過程中應(yīng)避免高溫,注意防潮;加速試驗表明,RLBN-TPT制劑批次間重現(xiàn)性較好,外觀、粒徑、含量和溶出度均無顯著變化且無藥物析出,有關(guān)物質(zhì)稍有增加,本品包裝及長期穩(wěn)定性還需進(jìn)一步考察。在藥代動力學(xué)實驗中,建立了LC-MS/MS法測定Wistar大鼠體內(nèi)拓?fù)涮婵笛帩舛?血漿中藥物濃度在1.0~200 ng/mL(r20.997)范圍,與峰面積比之間呈良好的線性關(guān)系,確定定量下限為1.0 ng/mL,方法回收率高,準(zhǔn)確度好,滿足Wistar大鼠體內(nèi)血藥濃度測定要求。以上市拓?fù)涮婵的z囊劑為參比制劑,比較了RLBN-TPT制劑在Wistar大鼠體內(nèi)的藥代動力學(xué)特征。結(jié)果表明,RLBNTPT制劑的Cmax和AUC0-∞分別是市售制劑的1.61倍和1.57倍(p0.05),顯著提高了藥物的口服生物利用度。為進(jìn)一步闡明反相脂質(zhì)納米粒的吸收途徑,比較了阻斷淋巴吸收途徑前后兩種制劑的體內(nèi)吸收行為,結(jié)果表明,阻斷后反相脂質(zhì)納米粒AUC顯著下降,下降至阻斷前的30%,市售制劑下降為阻斷前的63%,由此可以說明RLBN-TPT制劑通過增加藥物的淋巴吸收,從而提高了藥物的口服生物利用度。建立了BALB/c荷瘤裸鼠的HepG2實體瘤動物模型,結(jié)果顯示,RLBN-TPT組和上市制劑的抑瘤率分別為44.08%和16.3%(p0.05),通過TUNEL染色觀察腫瘤細(xì)胞凋亡、HE染色觀察腫瘤組織凋亡情況,并對腫瘤組織進(jìn)行病理學(xué)分析,結(jié)果表明,RLBN-TPT制劑組腫瘤細(xì)胞死亡面積更大,腫瘤組織大面積壞死。表明相比于市售制劑,RLBN-TPT制劑能夠更好地抑制腫瘤生長,具有更強抗腫瘤活性。在腸道刺激性試驗中,比較了RLBN-TPT制劑組與市售膠囊制劑組小鼠空腸的病變程度。由結(jié)果可知,服用市售制劑后小鼠腸黏膜層表面可見明顯水腫、脫失,以及血管充血,表明制劑對胃腸道具有較高的刺激性,而服用RLBN-TPT制劑后小鼠腸壁黏表層僅偶見水腫現(xiàn)象,其壁黏膜層及黏膜下、漿膜層、腸壁全層結(jié)構(gòu)清晰,微絨毛其柱狀上皮及杯狀細(xì)胞等細(xì)胞成分分布均勻,表明RLBNTPT制劑相較于市售膠囊制劑,降低了口服TPT腸道刺激性。綜上所述,RLBN-TPT制劑能夠顯著提高拓?fù)涮婵档姆�(wěn)定性并減少藥物對胃腸道刺激,促進(jìn)了藥物的淋巴吸收,提高了藥物的口服生物利用度。該制劑制備工藝簡單,不使用有機溶劑,輔料具有較高的生物相容性,應(yīng)用前景廣闊,其對淋巴轉(zhuǎn)運的促進(jìn)作用對淋巴密切相關(guān)的自身免疫等疾病的靶向治療具有重要意義。
[Abstract]:Topotecan (TPT) is a water-soluble semi-synthetic camptothecin (CPT) class of anticancer drugs, can specifically inhibit the function of topoisomerase I, hinder DNA replication. Topotecan is currently available in the form of topotecan injection freeze-dried powder injection and oral capsules. Compared with intravenous administration, oral TPT is effective, convenient, and has the characteristics of DNA replication. Low bone marrow inhibition, but because topotecan active lactone structure is affected by pH, in neutral and alkaline conditions, it is easy to hydrolyze to open ring to carboxylate configuration, thus losing activity, resulting in reduced bioavailability and antitumor activity, so the selection of appropriate drug carriers in the intestinal protection of its active configuration is particularly important. On the other hand, topotecan has strong gastrointestinal adverse reactions after oral administration. In view of the existing problems of TPT oral preparations, a new dosage form of topotecan reversed-phase lipid nanoparticles was prepared. The aim was to increase the stability of the drug in the digestive tract, reduce gastrointestinal mucosal irritation and improve the oral bioavailability of the drug. Usage. Single factor test and orthogonal test were used to screen the membrane materials, dosage and drug loading of reversed phase lipid nanoparticles. The oil solution of reversed phase lipid nanoparticles (RLBN-TPT) was prepared by freeze-drying process with medium chain triglyceride (MCT) as oil phase and the drug loading was 4mg/g. RLBN-TPT preparations were characterized by dynamic light scattering and transmission electron microscopy. The results showed that the particle size of RPL-TPT was about 60 nm and was uniform spherical. After dispersing in simulated intestinal fluid, the particle size and micro-morphology of RLBN-TPT preparations were observed. The results showed that uniform spherical lipid vesicles or emulsion droplets were formed after dispersing. A high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of topotecan in open-loop and closed-loop configurations. The stability of topotecan in artificial intestinal fluid was studied by measuring the release of topotecan reverse-phase lipid nanoparticles (RLBN-TPT) in simulated intestinal fluid, and the protective effect of topotecan in artificial intestinal fluid was investigated. APCELL PAK C18 MG II column (250 *4.6 mm, 5 micron), mobile phase A: 0.1% triethylamine (adjusting pH to 6.4 with glacial acetic acid) - B: acetonitrile [gradient: 0-8 min (88% A-60% A); 8-17 (60% A)], flow rate 1.0 mL/min, detection wavelength 360 nm, column temperature 55 The results show that the closed-loop configuration concentration is in the range of 0.25-5.00 UG mL-1, the concentration and peak area show a good line. Compared with topotecan aqueous solution, the stability of topotecan in reverse lipid nanoparticles in intestinal fluid was significantly enhanced, and the rate of formation of ring-opening configuration was significantly reduced (p0.05), indicating that reverse lipid nanoparticles have a certain protective effect on the form of drug-active lactones, effectively avoiding its short duration. A method for the determination of topotecan in RLBN-TPT preparations by HPLC was established. The results showed that the peak area and concentration of topotecan had a good linear relationship in the range of 0.125 micron g.mL-1 to 125 micron.mL-1, and the recovery of topotecan was between 98% and 102%. RSD is 0.67%. The method is accurate, precise, specific and sensitive, and can be used for the determination of topotecan in preparations. A method for the determination of topotecan in RLBN-TPT preparations by HPLC has been established. The methodological study shows that the method has high sensitivity, specificity and precision, and can be used for the determination of topotecan in preparations. Quality determination. The results of influencing factors showed that RLBN-TPT preparation was sensitive to humidity and heat, so high temperature should be avoided during storage, and moisture should be paid attention to. In the pharmacokinetic experiment, a LC-MS/MS method was established to determine the plasma concentration of topotecan in Wistar rats. The plasma concentration of topotecan in Wistar rats ranged from 1.0 ng/mL to 200 ng/mL (r20.997), and there was a good linear relationship between the plasma concentration and the peak area ratio. The pharmacokinetic characteristics of RLBN-TPT preparations in Wistar rats were compared with those of Topotecan capsules. The results showed that CMAx and AUC0-uuuuuuuuuuuuuuuuuuuuuuuu In order to further elucidate the absorption pathway of reversed-phase lipid nanoparticles, the in vivo absorption behaviors of the two preparations before and after blocking lymphatic absorption pathway were compared. The results showed that the AUC of reversed-phase lipid nanoparticles decreased significantly after blocking, to 30% before blocking, and 63% before blocking. Thus, the RLBN-TPT preparations were available. The results showed that the inhibition rates of RLBN-TPT group and marketed preparation were 44.08% and 16.3% (p0.05), respectively. The apoptosis of tumor cells was observed by TUNEL staining, and the apoptosis of tumor tissues was observed by HE staining. Pathological analysis of tumor tissues showed that RLBN-TPT preparation group had a larger area of tumor cell death and a larger area of tumor tissue necrosis. The results showed that the surface of intestinal mucosa was evidently edema, loss, and blood vessel congestion, indicating that the preparation had a high irritation to gastrointestinal tract, while the intestinal mucosal surface of mice treated with RLBN-TPT only occasionally showed edema, and the mucosal layer and submucosal layer, serosa of intestinal wall. In conclusion, RLBN-TPT preparation can significantly improve the stability of topotecan and reduce the gastrointestinal tract irritation and promote the development of drugs. Lymphatic absorption enhances the oral bioavailability of the drug. The preparation process is simple, does not use organic solvents, excipients have high biocompatibility and broad application prospects. Its promotion of lymphatic transport is of great significance for targeted treatment of lymph-related autoimmune diseases.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R943
本文編號：2206281
[Abstract]:Topotecan (TPT) is a water-soluble semi-synthetic camptothecin (CPT) class of anticancer drugs, can specifically inhibit the function of topoisomerase I, hinder DNA replication. Topotecan is currently available in the form of topotecan injection freeze-dried powder injection and oral capsules. Compared with intravenous administration, oral TPT is effective, convenient, and has the characteristics of DNA replication. Low bone marrow inhibition, but because topotecan active lactone structure is affected by pH, in neutral and alkaline conditions, it is easy to hydrolyze to open ring to carboxylate configuration, thus losing activity, resulting in reduced bioavailability and antitumor activity, so the selection of appropriate drug carriers in the intestinal protection of its active configuration is particularly important. On the other hand, topotecan has strong gastrointestinal adverse reactions after oral administration. In view of the existing problems of TPT oral preparations, a new dosage form of topotecan reversed-phase lipid nanoparticles was prepared. The aim was to increase the stability of the drug in the digestive tract, reduce gastrointestinal mucosal irritation and improve the oral bioavailability of the drug. Usage. Single factor test and orthogonal test were used to screen the membrane materials, dosage and drug loading of reversed phase lipid nanoparticles. The oil solution of reversed phase lipid nanoparticles (RLBN-TPT) was prepared by freeze-drying process with medium chain triglyceride (MCT) as oil phase and the drug loading was 4mg/g. RLBN-TPT preparations were characterized by dynamic light scattering and transmission electron microscopy. The results showed that the particle size of RPL-TPT was about 60 nm and was uniform spherical. After dispersing in simulated intestinal fluid, the particle size and micro-morphology of RLBN-TPT preparations were observed. The results showed that uniform spherical lipid vesicles or emulsion droplets were formed after dispersing. A high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of topotecan in open-loop and closed-loop configurations. The stability of topotecan in artificial intestinal fluid was studied by measuring the release of topotecan reverse-phase lipid nanoparticles (RLBN-TPT) in simulated intestinal fluid, and the protective effect of topotecan in artificial intestinal fluid was investigated. APCELL PAK C18 MG II column (250 *4.6 mm, 5 micron), mobile phase A: 0.1% triethylamine (adjusting pH to 6.4 with glacial acetic acid) - B: acetonitrile [gradient: 0-8 min (88% A-60% A); 8-17 (60% A)], flow rate 1.0 mL/min, detection wavelength 360 nm, column temperature 55 The results show that the closed-loop configuration concentration is in the range of 0.25-5.00 UG mL-1, the concentration and peak area show a good line. Compared with topotecan aqueous solution, the stability of topotecan in reverse lipid nanoparticles in intestinal fluid was significantly enhanced, and the rate of formation of ring-opening configuration was significantly reduced (p0.05), indicating that reverse lipid nanoparticles have a certain protective effect on the form of drug-active lactones, effectively avoiding its short duration. A method for the determination of topotecan in RLBN-TPT preparations by HPLC was established. The results showed that the peak area and concentration of topotecan had a good linear relationship in the range of 0.125 micron g.mL-1 to 125 micron.mL-1, and the recovery of topotecan was between 98% and 102%. RSD is 0.67%. The method is accurate, precise, specific and sensitive, and can be used for the determination of topotecan in preparations. A method for the determination of topotecan in RLBN-TPT preparations by HPLC has been established. The methodological study shows that the method has high sensitivity, specificity and precision, and can be used for the determination of topotecan in preparations. Quality determination. The results of influencing factors showed that RLBN-TPT preparation was sensitive to humidity and heat, so high temperature should be avoided during storage, and moisture should be paid attention to. In the pharmacokinetic experiment, a LC-MS/MS method was established to determine the plasma concentration of topotecan in Wistar rats. The plasma concentration of topotecan in Wistar rats ranged from 1.0 ng/mL to 200 ng/mL (r20.997), and there was a good linear relationship between the plasma concentration and the peak area ratio. The pharmacokinetic characteristics of RLBN-TPT preparations in Wistar rats were compared with those of Topotecan capsules. The results showed that CMAx and AUC0-uuuuuuuuuuuuuuuuuuuuuuuu In order to further elucidate the absorption pathway of reversed-phase lipid nanoparticles, the in vivo absorption behaviors of the two preparations before and after blocking lymphatic absorption pathway were compared. The results showed that the AUC of reversed-phase lipid nanoparticles decreased significantly after blocking, to 30% before blocking, and 63% before blocking. Thus, the RLBN-TPT preparations were available. The results showed that the inhibition rates of RLBN-TPT group and marketed preparation were 44.08% and 16.3% (p0.05), respectively. The apoptosis of tumor cells was observed by TUNEL staining, and the apoptosis of tumor tissues was observed by HE staining. Pathological analysis of tumor tissues showed that RLBN-TPT preparation group had a larger area of tumor cell death and a larger area of tumor tissue necrosis. The results showed that the surface of intestinal mucosa was evidently edema, loss, and blood vessel congestion, indicating that the preparation had a high irritation to gastrointestinal tract, while the intestinal mucosal surface of mice treated with RLBN-TPT only occasionally showed edema, and the mucosal layer and submucosal layer, serosa of intestinal wall. In conclusion, RLBN-TPT preparation can significantly improve the stability of topotecan and reduce the gastrointestinal tract irritation and promote the development of drugs. Lymphatic absorption enhances the oral bioavailability of the drug. The preparation process is simple, does not use organic solvents, excipients have high biocompatibility and broad application prospects. Its promotion of lymphatic transport is of great significance for targeted treatment of lymph-related autoimmune diseases.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R943
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，本文編號：2206281
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