Thesis (M.Sc)-Cairo nivsersity,2022.

Bibliography: p. 74-82.

Fungal keratitis (keratomycosis) is one of the most dangerous infections that makes damage to the eye and may cause blindness. It is a corneal inflammation of the eye that is caused by fungal invasion of the corneal surface commonly after trauma. The rate of incidence of fungal keratitis worldwide is between17% to 36%. This infection can affect all ages for both genders. The infection may be mild, moderate or severe. There are various types of fungi that are capable of colonizing human tissue. It may be yeast like Candida or filamentous like Trichosporon species and Aspergillus species.
Clotrimazole (CLZ), a synthetic imidazole derivative, is a broad spectrum antifungal agent. It is active against wide range of fungi, for example dermatophytes and Candida species. Like other imidazoles, it binds with the phospholipids of the fungi cell wall thus alters the membrane permeability, leading to leakage of essential elements from the cell like ions, metabolites and minerals such as potassium. The leakage of potassium outside the cell leads to acidification of the cell which causes activation of some enzymes that are responsible for destroying the cell. CLZ has been formulated in different conventional dosage forms like creams, gels, ovules and pessaries. Unfortunately, CLZ has poor water solubility which leads to decreasing of its therapeutic effect. Many trials have been made to increase CLZ water solubility like complexation with cyclodextrin as well as the development of different advanced delivery systems such as liposomes, niosomes and nanospheres.
There are a few data about the use of nanosized clotrimazole to treat ocular keratomycosis. This thesis was established to achieve this goal and the work in this thesis is divided into three chapters as follows:
Chapter I: Development and evaluation of clotrimazole loaded microemulsions.
Chapter II: Preparation and evaluation of clotimazole nano-cubosomal systems.
Chapter III: Microbiological studies of promising clotrimazole systems in rabbits.
 
Chapter I
Development and evaluation of clotrimazole loaded microemulsions
The aim of this work was to formulate Clotrimazole (CLZ), a water insoluble antifungal drug, into microemulsion for achieving an enhanced ocular delivery. For achieving this goal, the work in this chapter included the determination of the solubility of clotrimazole in different oils including oleic acid, paraffin oil and castor oil. After that the emulsification ability of different surfactants for the selected oil was determined. The tested surfactants were Cremophor EL, Solutol HS and Tween 80. Also, some co-surfactants were studied for their ability to increase emulsification ability of the surfactants. The studied co- surfactants were: propylene glycol, PEG 200, PEG 400 and Transcutol P. It was found that clotrimazole has highest solubility in oleic acid and the surfactants that achieved the best emulsification for this oil were Cremophor El and Solutol HS, so they were used for preparing the microemulsion systems. Regarding the best co-surfactant it was found that Transcutol P helped to increase the emulsification ability of the selected surfactants. When a transparent one-phase microemulsion system was developed, the physical state of the microemulsion was noted on the diagram of the pseudo-ternary components.
Every combination of surfactant and co-surfactant (having the ratios of 1:1, 1:2 and 1:3, respectively) was mixed with the selected oil at the ratios of (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9) respectively. Afterward, distilled water (aqueous phase) was added to the mixture and mixed by vortex till turbidity occurs. A pseudo ternary phase diagram was plotted using Chemix® software. Each component of the microemulsion was represented by one of the triangle’s apex. Based on the obtained diagrams, certain ratios were chosen to prepare the CLZ-loaded microemulsions.
CLZ-loaded microemulsions were formulated according to a 22 x 31 full factorial design which used for optimization of the microemulsions. In the current study, the type of surfactant, the ratio of surfactant to co-surfactant and the percentage of surfactant/ co-surfactant mixture (Smix) to the total preparation were selected as independent variables, whereas the dependent variables included droplet size (DS), polydispersity index (PDI), zeta potential (ZP) and release after 6 hours (Q6h).
Studying the droplet size of the prepared microemulsions, CLZ loaded microemulsions displayed DS in the nano range. The average DS of most of the prepared microemulsions was found to be within the reported values for microemulsions (20-200 nm). However, the obtained results demonstrated the relatively larger DS of F3, F7 and F8 (which have average values of 518, 967.5 and 949 nm, respectively). The analysis of the factorial design showed that the type of surfactant (X1) didn’t significantly influence the DS (P=0.0513). On the other hand, the % of Smix (X2) had a sig¬nificant effect on DS (P=0.0025). It was observed in the current study that the DS decreases upon increasing the % of Smix from 60% to 70 %.The ratio of surfactant to co-surfactant (X3), didn’t have effect on the DS (P=0.1813).
It was clear that F1, F2, F4, F6, F9, F10, F11and F12 have shown an acceptable PDI values (up to 0.7). On the other hand, F3, F5, F7 and F8 demonstrated larger PDI values. The factorial analysis showed that both the surfactant type (X1) and the ratio of surfactant to co-surfactant (X3) didn’t significantly influence the PDI (P=0.3993 and P=0.0814, respectively). However, % of Smix (X2) had big impact on PDI (P=0.0008). It was observed that PDI was reduced by increasing the Smix from 60% to 70%.
It was observed that the microemulsion systems had negative charge ranging between -22.00 mV to -33.30 mV. The presence of charge prevents particles aggregation. The factorial design showed that both the surfactant type (X1) and the ratio of surfactant to co-surfactant (X3) didn’t significantly influence ZP (P=0.6204 and P=0.1094, respectively). On the other hand, % of Smix (X2) had a significant effect on ZP (P=0.0001). It was observed that ZP decreases (as absolute value) upon increasing the % of Smix.
The in-vitro release of CLZ from several microemulsion systems over a period of 6 h was performed. The tested microemulsion systems showed sustained release profiles, F2 gave the highest release profile with 44.63% ± 20.84 of CLZ released after 6 h. The factorial analysis showed that the type of surfactant (X1) had a significant influence on Q6h (P= 0.0488). It was observed that release is affected by the type of surfactant as it increases with Cremophor EL and decreases with Solutol HS 15. On the other hand, the % of Smix (X2) and the ratio of surfactant: cosurfactant (X3) didn’t sig¬nificantly affect Q6h (P= 0.9477 and P= 0.4104).
Design Expert ® software was used to optimize the systems and select the optimum system. The selected microemulsion system was F4. The selected system (F4: containing oleic acid, Cremophor EL: Transcutol HP (1:1) and 70 % Smix) showed nanosized spherical droplets using transmission electron microscope (TEM), DS of (229.1 nm ± 0.9), PDI of (0.5±0.004), ZP of (-33.3 mV ± 0.98) and the amount released at 6 h (Q6h) was (37.6 % ± 4.47). The selected system was also coated with Chitosan coat. Generally, the obtained results confirmed that CLZ microemulsion could be promising for ocular drug delivery.
Chapter II
Preparation and evaluation of clotimazole
nano-cubosomal systems
The aim of this chapter was to prepare nano-cubosomal form of clotrimazole. CLZ-loaded nano-cubosomal systems were developed by emulsification of glycerol monooleate (GMO) with Pluronic F127 in aqueous phase. The influence of different variables (% of oily phase in the total system, % of Pluronic F127 in dispersed phase and sonication time) on the properties of the systems was investigated.
The mean size of all dispersions was in the nanometer (118.08 nm ±29.73 to 471.60 nm ±13.29). Factorial analysis showed that systems prepared using 10% of oily phase (C2, C4, C6 and C8) displayed significantly larger DS than systems prepared using 5% oily phase (C1, C3, C5 and C7) (P < 0.0001). However, the average DS of nano-cubosomal systems developed using 10% of Pluronic F127 in the dispersed phase (C1, C2, C5 and C6) did not significantly differ from those who have been developed using 20% of Pluronic F127 (C3, C4, C7 and C8) (P=0.4225). Moreover, statistical analysis revealed that nano-cubosomal systems subjected to 10 min sonication (C1-C4) showed a significantly smaller mean DS than that of the systems that were not sonicated (C5-C8) (P=0.0232).
Most of the prepared nano-cubosomal systems demonstrated acceptable PDI values. However, statistical analysis showed that the percentage of oily phase, the percentage of Pluronic F127 in the dispersed phase and sonication time did not have a big impact on the PDI of the prepared systems (P=0.478, P=0.848 and P=0.302, respectively).
It was observed that the prepared nano-cubosomal systems have a negative charge ranging between -23.6 mV ± 0.567 to -33.25 mV ± 0.07. Statistical analysis showed that systems prepared using 10% oily phase in the total system had a significantly higher ZP (as absolute value) in comparison with that of the systems prepared using 5% oily phase (P=0.0321). However, the percentage of PF127 in the dispersed phase didn’t have a big impact on ZP (P=0.0782). Moreover, statistical analysis revealed that ZP increased by increasing of the sonication time (P=0.00220).
Factorial design showed that systems prepared using 10% of oily phase relative to the total system (C2, C4, C6 and 8) had a significantly lower mean percentage of CLZ released at 6 hours (Q6h) in comparison with that of the systems prepared using 5% oily phase (C1, C3, C5 and C7) (P=0.0043). However, other system variables didn’t have a big impact on Q6h (P=0.9591 and P=0.393 for the percentage of Pluronic F127 and sonication time, respectively).
The optimum system (C7) (5% oily phase, 20% Pluronic F127 and 10 min sonication) showed nanosized nearly cubic particles with an average particle size of (182.60 nm ±17.68), polydispersity index of (0.30±0.01), zeta potential of (-26.50 mv ±1.13) and Q6h of (89.48 %±14.20). C7 was coated with chitosan lactate to increase the retention inside ocular tissues. Generally, results confirmed that CLZ chitosan coated nano-cubosomes could be promising for ocular drug delivery.
Chapter III
Microbiological studies of promising clotrimazole
systems in rabbits
In this chapter, selected clotrimazole systems, microemulsion system F4 and the chitosan coated form of F4 ( chapter I), cubosome system C7 and the chitosan coated form of (C7 chapter II) were subjected to in-vivo studies (Ocular irritation test and susceptibility test) in comparison with a drug suspension in rabbits. In addition to in-vivo histopathological studies.
A total of fifteen rabbits each of 2- 2.5 kg were involved in this study. They were checked to rule out any rabbits with symptoms of illness or inflammation of the eyes. Then they were separated into five groups of three rabbits each. The studied systems were exclusively applied to the right eye’s conjunctival sac, leaving the left eye as a control. Both eyes were evaluated for any signs redness, increase in lacrimation, conjunctival edema and hyperemia. The animals were examined after 2, 4, 6, 8, 24 and 48 h. The obtained results demonstrated that all the optimal systems and their chitosan-coated ones caused no irritation, no redness, no lacrimation or any sign of inflammation.
Regarding the susceptibility testing, the test organism, Candida Albicans was employed. The percentage of Candida Albicans growth inhibition was proportional to the drug residence time on the ocular surface after topical application. The rank of the fungal growth inhibition percentage (as evident from AUC 12h) of the tested CLZ systems compared to drug suspension is as follows: Chitosan coated C7 = Chitosan coated F4 > C7 >F4> drug suspension. It was clear that chitosan coating improved the residence of the systems on eye surface. To summarise, chitosan-coated microemulsion and cubosome systems could be a promising alternative to traditional eye drops, because of their long residence time and their sustained effect.
Histopathological studies have been performed to evaluate the tolerability of the selected systems. After one week of ocular application of different systems, the rabbits were killed under anesthesia and their eyeballs were separated, washed with normal saline and fixed by neutral buffered formalin for 72 h. The samples were then processed and imaged by a light microscope. After examination of (F4) and its coated form, there were no histopathological abnormalities in the tissues of the retina and the corneal region showed almost intact morphological features of different layers, however; mild widening and separation of collagen bundles were recorded only with chitosan-coated F4 system. After examination of (C7) and its coated form, There were no histopathological abnormalities in the tissues of the cornea and the different retinal layers showed almost intact morphological features of different layers, however; moderate edema in an inner plexiform layer, as well as significant loss of nuclear layer as well as ganglion cells layer, were recorded only in the chitosan-coated system. The chitosan cubosomal coated system requires further modification and investigation to prove its safety

المساعد (Smix ) بالنسبة لباقى مكونات المستحضر. و قد تمت دراسة أثر هذه المتغيرات على حجم الجزيئات ، مؤشر التشتت المتعدد(PDI )،جهد الزيتا (ZP) وكمية العقار المنطلقة بعد 6 ساعات. و قد أظهرت نتائج هذه الدراسة الاحصائية أن نوع المواد ذات النشاط السطحى له تأثير على حجم الجزيئات وكمية العقار المنطلقة بعد 6 ساعات، فى حين أن نسبة المواد ذات النشاط السطحى/ المواد ذات النشاط السطحى المساعد بالنسبة لباقى مكونات المستحضرأثرت على حجم الجزيئات و مؤشر التشتت المتعدد و جهد الزيتا.وبالنسبة إلى نسبة المواد ذات النشاط السطحى إلى المواد ذات النشاط السطحى المساعد فلم يكن له تأثير على كل العوامل تحت الدراسة. وقد تم استخدام برنامج Design Expert® لتحسين التركيبات واختيار الصيغة المثلى. وكانت الصيغة المختارة هى F4. أظهرت الصيغة المحددة (F4: تحتوي على % 70 من Smix و 1: 1 نسبة المواد ذات النشاط السطحى إلى المواد ذات النشاط السطحى المساعد والكريموفور- إى ل هو المادة ذات النشاط السطحى). كما تم إضافة طبقة خارجية من الكيتوزان للصيغة المحددة من أجل زيادة وقت الالتصاق بأنسجة العين. بشكل عام، أكدت النتائج التي تم الحصول عليها أن المستحلبات الدقيقة المصنعة من الكلوتريمازول يمكن أن تكون واعدة لاستخدام الدواء فى العين. وبناء على ما سبق فقد تم اختيار الصيغة (F4) لمزيد من دراسات التقييم الحيوى على أعين الأرانب و ذلك فى الفصل الثالث. الفصل الثاني تحضير وتقييم أنظمة كيوبوزومية محملة بالكلوتريمازول في هذا الجزء من الدراسة تم تحضير عقار الكلوتريمازول في صورة أنظمة كيوبوزومية نانوية (nano-cubosomal systems) بواسطة استحلاب الجليسرول مونوأوليات مع ، بلورونيك- إف-127 في الماء المقطر.وقد تم دراسة تأثير نسبة المحتوى الزيتي بالنسبة لكمية المستحضر، نسبة مادة بلورونيك- إف-127 في المحتوى المشتت و كذلك مدة تعرُّض الأنظمة المحضرة للموجات فوق الصوتية على كل من حجم الجزيئات ، مؤشر التشتت المتعدد(PDI )،جهد الزيتا (ZP) وكمية العقار المنطلقة بعد 6 ساعات. وقد أظهرت نتائج هذه الدراسة الاحصائية أن نسبة المحتوى الزيتي بالنسبة لكمية المستحضر له تأثير على حجم الجزيئات(DS) و جهد الزيتا (ZP) وكمية العقار المنطلقة بعد 6 ساعات بينما لم تظهر نسبة بلورونيك- إف-127 اى تأثير على عامل تحت الدراسة .وقد أظهرت مدة وقت تعرض الأنظمة المحضرة للموجات فوق الصوتية تأثير على حجم الجزيئات وجهد الزيتا (ZP). وقد تم استخدام برنامج Design Expert ® لاختيار الصيغة المثلى وكانت التركيبة المختارة هى C7. (C7: تحتوي على 5% من المحتوى الزيتي بالنسبة لكمية المستحضر و 20% بلورونيك- إف-127 و خضعت إلى 10 دقائق من الموجات الفوق الصوتية). كما تم إضافة طبقة خارجية من الكيتوزان للصيغة المحددة من أجل زيادة وقت الالتصاق بأنسجة العين. بشكل عام، أكدت النتائج التي تم الحصول عليها أن الأنظمة الكيوبوزومية المحملة بعقار الكلوتريمازول يمكن أن تكون واعدة لاستخدام الدواء فى العين. بناءً على ما سبق فقد تم اختيار الصيغة (C7) لمزيد من دراسات التقييم الحيوى على أعين الأرانب. الفصل الثالث دراسات ميكروبيولوجية على أنظمة الكلوتريمازول الواعدة فى الأرانب في هذا الفصل تم اختيارصيغتين من الكلوتريمازول (F4, C7 ) من الفصلين الأول و الثاني وكذلك أشكالهم المغطاة بطبقة خارجية من الكيتوزان من أجل القيام بدراسات التقييم الحيوى فى الأرانب (اختبار تهيج العين وإجراء اختبار الحساسية ) بالمقارنة مع محلول معلق من الدواء. تم وضع الصيغتين تحت الدراسة على كيس الملتحمة للعين اليمنى، وترك العين اليسرى كضابط . تم تقييم كلتا العينين لأي علامات احمرار، وزيادة في الإثارة، والتهابات الملتحمة، وفرط الدم. لقد فحصت الحيوانات بعد 2 و 4 و 6 و 8 و 24 و 48 ساعة. أظهرت النتائج التي تم الحصول عليها أن جميع الصيغ المثلى وغير المطلية بالكيتوزان لم تشعر بالتهيج، لا احمرار، زيادة دموع ، أو أي علامة على التهاب الملتحمة. فيما يتعلق باختبار الحساسية، كانت Candida Albicans هو الفطر الذى ُاُستخدم لإجراء الاختبارات . كانت النسبة المئوية لتثبيط نمو الفطريات يتناسب مع وقت الدواء على سطح العين بعد الاستخدام الموضعي. كانت النسبة المئوية لعينات المستحلب الدقيق على النحو التالي: C7 المغطاة بطبقة خارجية من الكيتوزان = F4 المغطاة بطبقة خارجية من الكيتوزان > < (F4) < (C7) معلق من الدواء. كان من الواضح أن الصياغات المحضرة نجحت في الحفاظ على النشاط المضاد للفطريات على مدى فترة أطول بالمقارنة مع معلق الدواء. أيضا، إضافة طبقة خارجية من الكيتوزان للعينات عمل على تحسين التصاقها على سطح العين. إجمالًا ، يمكن أن تكون التركيبات المحضرة المغطاة من الخارج بالكيتوزان بديلا لقطرات العين التقليدية، بسبب وقت التصاقها الطويل وتأثيرها المستمر.

Issues also as CD.

Text in English and abstract in Arabic & English.