Preparation and evaluation of ophthalmic liposomal voriconazole formulation by thin film hydration method

황금길 Graduate School, Yonsei University 2018 국내석사

1. Introduction Fungal keratitis is one of infectious corneal diseases and is an increasing disease in developing countries. And the incidence is increasing in developed countries due to increased use of contact lenses. To treat this, antifungal agents are used, typically natamycin and amphotericin B. However, its use is limited because of the narrow range of antifungal agents and low permeability. Voriconazole, a second-generation azole antifungal agent with a broad range of antifungal agents, is under investigation. The limitation of Voriconazole is that it has low water solubility and poor cornea permeability. Liposome as nano-carrier can be used to overcome this problem. Voriconazole can be encapsulated between phospholipids of liposome to enhance permeability while increasing solubility. There are various ways of making liposomes. Among the various methods of making liposomes, liposomes were prepared by the commonly used thin film hydration method. Therefore, the goal of this paper is to develop a liposomal formulation of voriconazole with broad spectrum antifungal activity, which is increased water solubility and permeability, thereby increasing the BA of voriconazole and ultimately increasing therapeutic efficiency. 2. Preparation of liposome We prepared liposome by thin film hydration. Formulations of Increasing the organic solvent amount and molar ratio of HSPC were prepared. And we also prepared formulations containing a mPEG-DSPE, SA for positively charged liposome, and DP for negatively charged liposome. Cholesterol was added to find out effect of cholesterol to liposome. 3. Evaluation of liposome Organic solvent amount did not affect to size, zeta potential, assay%, EE%. Increasing molar ratio of HSPC affect to the size, PDI, and pH. Cholesterol did not affect to liposome size, PDI and assay%, EE%, also. Formulation with SPC was viscous and size was largest. mPEG-DSPE makes zeta potetial of liposome to negative charge but stearylamine makes zeta potential to positive. Dicetylphosphate makes liposome acidic but stearylamine makes liposome basic. Voriconazole assay % was almost 100% and EE% was almost 94% in all formulation. Specially formulation with mPEG-DSPE showed 99% of EE%. In the DSC data, voriconazole peak and cholesterol peak was disappeared in all formulations. In vitro release data described that all liposome formulation has higher dissolution rate than 1% voriconazole suspension. Formulation with mPEG-DSPE, Chol shows the higher dissolution rate. And permeation study with bovine cornea described that permeation rate followed the zeta potential. Formulation 12, and 15 have higher permeation rate and stearylamine formulation with positive zeta potential show lowest permeation rate. 진균 각막염은 감염성 각막 질환 중 하나이며 개발 도상국에서 증가하는 질병이다. 그리고 콘택트 렌즈의 사용 증가로 인해 선진국에서는 그 빈도가 증가하고 있다. 이를 치료하기 위해 일반적으로 natamycin과 amphotericin B와 같은 항진균제가 사용된다. 그러나 이들 항균제들은 항균 범위가 좁고 각막 침투성이 낮기 때문에 사용이 제한됩니다. 보리코나졸 (Voriconazole)은 다양한 종류의 항진균 범위를 가진 2 세대 azole계 항진균제이다. 보리코나졸의 사용 제한점은 용해도가 낮고 수분 안정성이 낮다는 것이다. 이 문제를 극복하기 위해 나노 운반체 인 리포좀을 사용할 수 있다. 보리코나졸은 리포솜의 인지질 사이에 encapsulation 되어 안정성이 높아지고 용해도도 높아질 수 있다. 리포솜을 제조하는 다양한 방법 중에서, 일반적으로 사용되는 thin film hydration법으로 리포좀을 제조 하였다. 따라서 본 연구의 목적은 용해도 및 투과성이 증가 된 광범위한 항진균 활성을 갖는 보리 코나 졸의 리포좀 제형을 개발함으로써 보리 코나 졸의 BA를 증가시키고 궁극적으로 치료 효율을 증가시키는 것이다. 2. 리포솜의 제조 Thin film hydration법을 리포솜을 만들었다. HSPC의 유기 용매량 및 몰비를 증가시키는 표뮬레이션을 개발 하였다. 그리고 우리는 또한 mPEG-DSPE, 양전하를 띤 리포솜을 위한 SA 및 음전하를 띠는 리포솜을 위한 DP를 함유하는 제제를 만들었다. 콜레스테롤이 리포솜에 대한 콜레스테롤의 효과를 알아 내기 위해 첨가되었습니다. 3. 리포좀의 평가 유기 용매의 양은 particle mean size, zeta potential, assay%, EE %에는 영향을 미치지 않았다. HSPC의 증가하는 몰 비율은 particle mean size, PDI, pH에 영향을 미친다. 콜레스테롤은 리포좀 mean size, PDI 및 assay%, EE%에도 영향을 미치지 않았다. SPC를 사용한 제제는 점성이 크고 입자 크기가 가장 컸다. mPEG-DSPE는 리간드의 제타 전위를 음전하로하지만 stearylamine은 리포솜의 zeta potential을 양전하로 만든다. dicetylphosphate는 리포좀을 산성으로 만들지만 stearylamine은 리포좀을 염기성으로 만든다. Voriconazole assay%은 거의 100 % 였고 EE %는 거의 모든 포뮬레이션에서 거의 94 %였다. 특히 mPEG-DSPE를 사용한 제제는 99 %의 EE %를 나타냈다. DSC 데이터에서 모든 포뮬레이션에서 보리코나졸 peak와 콜레스테롤 peak가 사라졌다. Drug release study는 모든 리포좀 제형이 1 % 보리코나졸 현탁액보다 높은 용출률을 갖는 것을 의미했다. Chol과 mPEG-DSPE을 함유한 포뮬레이션은 더 높은 용출률을 나타냈다. 소의 각막을 이용한 permeability study에서 permeability rate가 zeta potential과 비례한다고 할 수 있다. 포뮬레이션 12, 포뮬레이션 15는 보다 높은 permeability rate를 가지며, 양의 zeta potential을 갖는 stearylamine 포뮬레이션은 가장 낮은 투과 속도를 나타냈다.

웅담·우황약침액의 안점막자극실험 및 다종의 각막염 유발균에 미치는 영향

한나영 상지대학교 대학원 2010 국내석사

감염성 각막염을 치료하기 위한 점안제로 웅담·우황약침액을 활용하기 위하여 안점막자극에 미치는 영향을 관찰하고자, 식품의약품안전청 고시 제2005-60호 “의약품 등의 독성시험기준(2005. 10. 21. 개정)”에 따라 토끼를 이용한 안점막자극 시험을 실행하였고, 각막염 유발균 Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans에 미치는 항균효과를 알아보기 위하여 억제환 및 최소 성장 억제 농도 측정 시험을 통하여 다음과 같은 결과를 얻었다. 1. 실험에 이용된 9마리의 토끼 모두에게서 웅담·우황약침액에 의해 발생된 임상증상, 체중변화의 이상은 관찰되지 않았다. 2. 웅담·우황약침액 투여 후 1, 2, 3, 4 및 7일에 각각 각막, 홍채, 결막에 검액에 의해 유발된 안구병변은 관찰되지 않았다. 3. 웅담·우황약침액은 Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans에 대하여 50 ㎕ 전농도에서 억제효과가 나타나지 않았다. 이상의 결과로 보아 웅담·우황약침액은 토끼의 안점막에 있어서 어떠한 안구병변도 유발하지 않는 무독성, 무자극성 약물로 판단되었다. 그러나, 각막염 유발 세균 및 진균 6종에 대하여 항균효과가 없다는 것을 알 수 있었다. 따라서 감염성 각막염 치료에 활용할수 있는 안정성과 함께 높은 향균효과를 가진 한방 점안제에 대한 다각적 연구가 필요할 것으로 사료된다. ABSTRACT The Experimental study on the effect of Fel Ursi & Bovis Calculus Pharmacopuncture solution in eye irritation test and in bacterial species which cause Keratitis Na-Young Han Dept. of Korean Medicine Graduate School, Sangji University (Directed by Hyung-sik Seo, O.M.D., Ph.D.) Objective : This experimental study was performed to investigate the safety of eye irritation test of rabbits and the validity of antibacterial test using Fel Ursi & Bovis Calculus pharmacopuncture solution manufactured by extraction of alcohol and water for identifying the use of it as eyedrops. Methods : 1. The eye irritation test of the Fel Ursi & Bovis Calculus pharmacopuncture solution performed on left eye of 9 white rabbits which has no ophthalmic disease. The test including irritaion of cornea(opacity), iris(values) and conjunctiva(redness, chemosis, and discharge) was observed at 1, 2, 3, 4 & 7day, according to the Regulation of Korea Food & Drug Administration(2005. 10. 21, KFDA 2005-60). 2. Measure the size of inhibition zone and MIC(Minimum Inhibition Concentration) after administering Fel Ursi & Bovis Calculus pharmacopuncture solution on bacterial species, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum and Candida albicans, which cause Keratitis. Administering Cravit(Levofloxacin medicine) on bacterial species also performed to compare the anti-bacterial potency of this material, measured by using the size of inhibition zone. Results : 1. There was no biological problem such as change of weight, diet amount, moving condition, after Soyeom pharmacopuncture solution was administered in the left eye of the rabbits. 2. There was no eye irritation of the cornea, iris and conjunctiva was observed at 1, 2, 3, 4 & 7day, after observing the Soyeom pharmacopuncture solution was medicated in the left eye of the rabbits. 3. After administering Fel Ursi & Bovis Calculus pharmacopuncture solution on bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans), there was no response to MIC and there was no anti-bacterial potency also. Conclusions : This study suggests that Fel Ursi & Bovis Calculus pharmacopuncture solution is non-toxic and non-irritant because there was no eye irritation in rabbits, but no anti-bacterial effects was shown on bacterial species which cause Keratitis. These study result recommends that we need to research more on herbal medicines of eye drop which is safe and antibiotic for Keratitis.

소염약침액의 안점막자극실험 및 다종의 각막염 유발균에 미치는 영향

강은교 尙志大學校 大學院 2009 국내석사

Objective : This experimental study was performed to investigate the safety of Soyeom Pharmacopunture solution manufactured by extraction of alcohol and water. To identify the use of it as eyedrops, the eye irritation test of rabbits and antibacterial test of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, and Candida albicans was performed. Methods : 1. The eye irritation test of this material was performed according to the Regulation of Korea Food & Drug Administration(2005. 10. 21, KFDA 2005-60). After Soyeom pharmacopuncture solution was administered in the left eye of the rabbits, eye irritation of the cornea, iris and conjunctiva was observed at 1, 2, 3, 4 & 7day. 2. After administering Soyeom Pharmacopuncture solution on bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans) which cause Keratitis, MIC(Minimum Inhibition Concentration) and the size of inhibition zone were measured. Anti-bacterial potency was also measured using the size of inhibition zone. Results : 1. After Soyeom pharmacopuncture solution was administered in the left eye of the rabbits, it was found that none of nine rabbits have abnormal signs and weight changes. 2. After Soyeom pharmacopuncture solution was medicated in the left eye of the rabbits, no eye irritation of the cornea, iris and conjunctiva was observed at 1, 2, 3, 4 & 7day. 3. There was no response to MIC on bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans) after Soyeom pharmacopuncture solution was medicated. Conclusions : The present study suggests that Soyeom pharmacopuncture solution is a non-toxic and non-irritant medicine, which does not cause eye irritation in rabbits, but dosen't have anti-bacterial effects on bacterial species which cause Keratitis. These study result recommends that more research on other herbal medicines of eye drop for Keratitis are required.