達(dá)托霉素和克拉霉素共載藥脂質(zhì)體的研究
發(fā)布時(shí)間：2019-05-24 01:48
【摘要】：耐甲氧西林金黃色葡萄球菌(MRSA)引起的感染嚴(yán)重影響了公眾的健康,甚至是已用某些抗生素治療過MRSA引起的感染還會(huì)導(dǎo)致高的死亡率,隨著病原體耐藥防控難度的持續(xù)增大,除了不斷研發(fā)新型抗生素之外,對已有抗生素進(jìn)行新制劑創(chuàng)制與聯(lián)合給藥具有同等的重要性。本研究旨在評估達(dá)托霉素和克拉霉素聯(lián)用對耐甲氧西林金黃色葡萄球菌(MRSA)感染的協(xié)同作用效果。 本文采用聚乙二醇修飾的脂質(zhì)體作為載體,考察并系統(tǒng)評價(jià)經(jīng)優(yōu)化制備的達(dá)托霉素、克拉霉素共載藥脂質(zhì)體(PL[CD])抗耐甲氧西林金葡菌(MRSA252)全身感染。通過優(yōu)化制備得到的達(dá)托霉素、克拉霉素共載藥脂質(zhì)體(PL[CD])平均粒徑(98.2±2.21)nm,多分散系數(shù)為0.251,包封率分別為(92.94±1.21,94.71±1.37)%,載藥量為(8.45±0.11,0.29±0.043)%,粒徑大小均一,并具有良好的穩(wěn)定性。在臨床劑量下,體內(nèi)外評價(jià)結(jié)果顯示,達(dá)托霉素和克拉霉素共載藥脂質(zhì)體(PL[CD])與達(dá)托霉素脂質(zhì)體及克拉霉素脂質(zhì)體制劑相比,對MRSA252有更持久的抑制效果,并能顯著提高耐甲氧西林金葡菌全身感染模型小鼠的存活率上述研究結(jié)果表明,達(dá)托霉素和克拉霉素共載藥脂質(zhì)體在體外能夠顯著增強(qiáng)對抗MRSA的活性,在體內(nèi)能顯著減少M(fèi)RSA的定植量并能增加宿主的生存率。因此,可為今后臨床應(yīng)用提供相關(guān)的依據(jù)。 本課題的主要研究內(nèi)容如下： (1)達(dá)托霉素、克拉霉素體外協(xié)同優(yōu)選方案 通過棋盤法對達(dá)托霉素、克拉霉素在體外進(jìn)行聯(lián)合,通過計(jì)算,聯(lián)合抑菌指數(shù)(FIC)為0.5,可初步判定達(dá)托霉素與克拉霉素體外聯(lián)用有協(xié)同作用。 (2)單藥脂質(zhì)體的制備及體外協(xié)同方案優(yōu)化 克拉霉素高效液相色譜法在45℃條件下,使用C18色譜分離柱,流速為0.7mL/min,流動(dòng)相是含有0.1%三氟乙酸的乙腈與3.5mM的HC1調(diào)pH至3.5±0.2的0.033nol/L KH2PO4(40/60,v/v),檢測波長為205nm；達(dá)托霉素的高效液相色譜法在35℃條件下,流速為1mL/min,流動(dòng)相是乙腈與0.5%的NH4H2PO4(36/64,v/v),檢測波長為221nrn。 通過考察制備達(dá)托霉素長循環(huán)脂質(zhì)體的幾個(gè)影響因素：氫化卵磷脂(HSPC)與膽固醇的摩爾比、脂藥質(zhì)量比,最終得出制備達(dá)托霉素長循環(huán)脂質(zhì)體的最優(yōu)處方,就能得到穩(wěn)定的脂質(zhì)體劑型。在4℃的冰箱中保存2個(gè)月后幾乎無泄漏、無沉淀現(xiàn)象發(fā)生。成功制備的克拉霉素、達(dá)托霉素單藥脂質(zhì)體聯(lián)合抑菌指數(shù)主要采用棋盤法來測定,得到PL[C]與PL[D]最優(yōu)化的比率為32：1,此比值用于后續(xù)制備共載藥脂質(zhì)體。 (3)共載藥脂質(zhì)體的制備與評價(jià) 通過優(yōu)化制備得到的達(dá)托霉素、克拉霉素共載藥脂質(zhì)體平均粒徑(98.2+2.21)nm,多分散系數(shù)為0.251,包封率分別為(92.94±1.21,94.71±1.37)%,載藥量為(8.45±0.11,0.29±0.043)%,粒徑大小均一,并具有良好的穩(wěn)定性。 體外抗耐甲氧西林金葡菌實(shí)驗(yàn)主要考察達(dá)托霉素三個(gè)制劑：克拉霉素脂質(zhì)體(PL[C])、達(dá)托霉素脂質(zhì)體(PL[D])、達(dá)托霉素、克拉霉素共載藥脂質(zhì)體(PL[CD])對MRSA252生長的影響,結(jié)果表明,PL[CD]對MRSA252的抑制效果更持久。生物膜的定性和定量結(jié)果的分析表明,PL(CD)比PL[C], PL[D]抑制MRSA的效果更強(qiáng)。 通過比較PL[C]、PL[D]、PL[CD]對MRSA252致全身感染KM小鼠的治療效果,結(jié)果顯示,臨床劑量下,達(dá)托霉素長循環(huán)脂質(zhì)體能使感染KM小鼠的存活率在觀察期(7天)提高到90%。安全性評價(jià)結(jié)果表明共載藥脂質(zhì)體未對KM小鼠沒有明顯毒性。
[Abstract]:The infection caused by methicillin-resistant Staphylococcus aureus (MRSA) seriously affects the health of the public, and even the infection that has been caused by the treatment of MRSA with certain antibiotics can also lead to high mortality, and as the prevention and control difficulty of the pathogen increases continuously, In addition to the constant development of new types of antibiotics, the new formulation of existing antibiotics has the same importance as the combination. The purpose of this study was to assess the synergistic effect of daptomycin and clarithromycin on the infection of methicillin-resistant Staphylococcus aureus (MRSA). In this paper, polyethylene glycol modified liposomes were used as carrier to study and evaluate the systemic sense of daptomycin and clarithromycin co-carrier liposome (PL[CD]) against methicillin-resistant Staphylococcus aureus (MRSA252). The average particle size (98.2-2.21) nm, the multi-dispersion coefficient of 0.251 and the encapsulation rate of the daptomycin and the clarithromycin co-carrier liposome (PL[CD]) were (92.94-1.21, 94.71-1.37)%, (8.45-0.11, 0.29-0.043)% and the particle-size-size of the liposome (PL[CD]). I. It has good stability. Sex. In the clinical dose, the in-vivo evaluation results show that the daptomycin and clarithromycin co-carrier liposomes (PL[CD]) have a more durable inhibitory effect on the MRSA252 compared to the daptomycin liposome and the clarithromycin liposome formulation. The results of the study show that the co-carrier of daptomycin and clarithromycin can significantly enhance the activity against MRSA in vitro, and can significantly reduce the colonization of MRSA in vivo and increase the survival of the host. Rate. Therefore, it can be relevant to the clinical application in the future According to the main research of the subject The volume is as follows: (1) in vitro of daptomycin and clarithromycin in a synergistic prefer scheme, daptomycin and clarithromycin are combined in vitro by a checkerboard method, 0.5. It can be used to determine the in vitro of daptomycin and clarithromycin in vitro. co-acting. (2) The preparation of a single-agent liposome The preparation and in vitro synergistic scheme optimizes the clarithromycin high performance liquid chromatography to use a C18 chromatographic separation column under the condition of 45 DEG C, the flow rate is 0.7 mL/ min, the mobile phase is acetonitrile with 0.1% trifluoroacetic acid and 3.5 mM HC1 to adjust the pH to 0.033 nol/ L KH2PO4 of 3.5 to 0.2 (40/60, v/ v) with a detection wavelength of 205 nm; a high performance liquid chromatography of daptomycin at a flow rate of 1 mL/ min at 35 C and a mobile phase of acetonitrile and 0.5% NH4H2PO4 (36/64, v/ v), The detection wavelength was 221 nrn. Several factors influencing the preparation of daptomycin long-cycle liposomes were investigated: the molar ratio of hydrogenated lecithin (HSPC) to cholesterol, the mass ratio of lipid, and finally the optimal formulation of the preparation of daptomycin long-cycle liposomes was obtained. and a stable liposome dosage form can be obtained, and after two months are stored in a refrigerator at 4 DEG C, The combined antibacterial index of clarithromycin and daptomycin as a single agent was determined by a checkerboard method, and the ratio of PL[C] to PL[D] was 32:1. The value is used for subsequent preparation of the co-carrier liposome. (3) The preparation and evaluation of the co-carrier liposomes were optimized to prepare the daptomycin, the clarithromycin co-carrier liposome average particle size (98.2 + 2.21) nm, the polydispersity index of 0.251, the encapsulation rate (92.94% 1.21, 94.71% 1.37)%, and the drug loading (8.45%). 0.11, 0.29 (0.043)% The anti-methicillin-resistant Staphylococcus aureus in vitro mainly studied the three preparations of daptomycin: clarithromycin liposome (PL[C]), daptomycin liposome (PL[D]), daptomycin, and clarithromycin co-carrier liposome (PL[CD]). ) Effect on the growth of MRSA252, the results show that PL[ CD] The inhibition of MRSA252 is more durable. The analysis of the qualitative and quantitative results of the biofilm shows that the PL (CD) ratio PL[ [C], PL[D] inhibited the efficacy of MRSA. By comparing PL[C], PL[D], and PL[CD] to the therapeutic effect of MRSA252 on the whole body infected KM mice, the results showed that the long circulating liposomes of daptomycin can make the infection K The survival of M mice increased to 90% in the observation period (7 days). The safety evaluation junction
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943
本文編號(hào)：2484440
[Abstract]:The infection caused by methicillin-resistant Staphylococcus aureus (MRSA) seriously affects the health of the public, and even the infection that has been caused by the treatment of MRSA with certain antibiotics can also lead to high mortality, and as the prevention and control difficulty of the pathogen increases continuously, In addition to the constant development of new types of antibiotics, the new formulation of existing antibiotics has the same importance as the combination. The purpose of this study was to assess the synergistic effect of daptomycin and clarithromycin on the infection of methicillin-resistant Staphylococcus aureus (MRSA). In this paper, polyethylene glycol modified liposomes were used as carrier to study and evaluate the systemic sense of daptomycin and clarithromycin co-carrier liposome (PL[CD]) against methicillin-resistant Staphylococcus aureus (MRSA252). The average particle size (98.2-2.21) nm, the multi-dispersion coefficient of 0.251 and the encapsulation rate of the daptomycin and the clarithromycin co-carrier liposome (PL[CD]) were (92.94-1.21, 94.71-1.37)%, (8.45-0.11, 0.29-0.043)% and the particle-size-size of the liposome (PL[CD]). I. It has good stability. Sex. In the clinical dose, the in-vivo evaluation results show that the daptomycin and clarithromycin co-carrier liposomes (PL[CD]) have a more durable inhibitory effect on the MRSA252 compared to the daptomycin liposome and the clarithromycin liposome formulation. The results of the study show that the co-carrier of daptomycin and clarithromycin can significantly enhance the activity against MRSA in vitro, and can significantly reduce the colonization of MRSA in vivo and increase the survival of the host. Rate. Therefore, it can be relevant to the clinical application in the future According to the main research of the subject The volume is as follows: (1) in vitro of daptomycin and clarithromycin in a synergistic prefer scheme, daptomycin and clarithromycin are combined in vitro by a checkerboard method, 0.5. It can be used to determine the in vitro of daptomycin and clarithromycin in vitro. co-acting. (2) The preparation of a single-agent liposome The preparation and in vitro synergistic scheme optimizes the clarithromycin high performance liquid chromatography to use a C18 chromatographic separation column under the condition of 45 DEG C, the flow rate is 0.7 mL/ min, the mobile phase is acetonitrile with 0.1% trifluoroacetic acid and 3.5 mM HC1 to adjust the pH to 0.033 nol/ L KH2PO4 of 3.5 to 0.2 (40/60, v/ v) with a detection wavelength of 205 nm; a high performance liquid chromatography of daptomycin at a flow rate of 1 mL/ min at 35 C and a mobile phase of acetonitrile and 0.5% NH4H2PO4 (36/64, v/ v), The detection wavelength was 221 nrn. Several factors influencing the preparation of daptomycin long-cycle liposomes were investigated: the molar ratio of hydrogenated lecithin (HSPC) to cholesterol, the mass ratio of lipid, and finally the optimal formulation of the preparation of daptomycin long-cycle liposomes was obtained. and a stable liposome dosage form can be obtained, and after two months are stored in a refrigerator at 4 DEG C, The combined antibacterial index of clarithromycin and daptomycin as a single agent was determined by a checkerboard method, and the ratio of PL[C] to PL[D] was 32:1. The value is used for subsequent preparation of the co-carrier liposome. (3) The preparation and evaluation of the co-carrier liposomes were optimized to prepare the daptomycin, the clarithromycin co-carrier liposome average particle size (98.2 + 2.21) nm, the polydispersity index of 0.251, the encapsulation rate (92.94% 1.21, 94.71% 1.37)%, and the drug loading (8.45%). 0.11, 0.29 (0.043)% The anti-methicillin-resistant Staphylococcus aureus in vitro mainly studied the three preparations of daptomycin: clarithromycin liposome (PL[C]), daptomycin liposome (PL[D]), daptomycin, and clarithromycin co-carrier liposome (PL[CD]). ) Effect on the growth of MRSA252, the results show that PL[ CD] The inhibition of MRSA252 is more durable. The analysis of the qualitative and quantitative results of the biofilm shows that the PL (CD) ratio PL[ [C], PL[D] inhibited the efficacy of MRSA. By comparing PL[C], PL[D], and PL[CD] to the therapeutic effect of MRSA252 on the whole body infected KM mice, the results showed that the long circulating liposomes of daptomycin can make the infection K The survival of M mice increased to 90% in the observation period (7 days). The safety evaluation junction
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943
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