الفهرس 
Introduction
Part I: Materials And MethodsOnly 14 pages are availabe for public view
 |  | from | 273 |  |  |
| | | from | 273 | | |
Abstract
Drugs with antifungal activity were first identified in 1939 when griseofulvin was introduced [4-6]. from then, several agents were introduced until azoles appeared in 1969 [4, 14]. Azoles are the most widely used and studied category of antifungals [4]. This is because they are highly effective with low toxicity and low risk of secondary side effects, in addition to possessing immunomodulatory activity [1-3, 22, 38, 39]. Azole antifungals are either imidazoles or triazoles [1, 36]. Both types have the same mechanism of action, as well as, the same range of use and antifungal spectrum [1, 38]. They can inhibit the demethylation of sterol’s carbon-14 in the fungal cell walls by inhibition of 14 alpha- sterol demethylase (lanosterol demethylase), enzyme in the fungi [12, 36, 40-43]. Thus, they interfere with the normal biosynthesis of ergosterol, by hindering the conversion of lanosterol to ergosterol modifying its biochemical composition, and leading to accumulation of 14 alpha-methyl sterols [4, 21, 36, 38, 41-43]. This inhibits fungal cell growth, and disrupt the hormone-like function of ergosterol [4, 12, 21, 36, 38, 40-43]. Also, they interfere with cell division and replication and eventually cause cell death [1, 20, 39, 44-47]. Hence, they can disrupt both the structure and several of the functions of the fungal membrane including nutrient transport and chitin synthesis [4, 12, 40, 43]. This results in loss of membrane integrity and disruption of the osmotic balance of the cell membrane, leading to the leakage of intracellular ions, the same kind of damage caused by polyenes [36]. 14 alpha demethylase is also involved in cholesterol synthesis in mammals, however they have a greater affinity for the fungal isoenzyme than its mammalian counterpart [4, 41]. Systemic triazoles are metabolized slower than imidazoles and exert less effect on human sterol synthesis [1, 3, 22, 38, 39, 43]. Triazoles also have a better safety profile compared to imidazoles, due to their higher affinity for the fungal enzyme [4].
Both of the selected drugs, itraconazole and voriconazole, belong to the triazole category of azole antifungals [1-3, 20, 22, 30-32, 37, 45, 48-56]. They are both highly effective against a wide range of clinically important fungi [44, 60, 94, 95, 104, 105, 111, 113, 114][4, 19, 58[48, 49, 57]. Both drugs are poorly water soluble (BCS class II) [57, 96, 121]. Their aqueous solubility is too low to be consistent with complete absorption due to poor dissolution that results in bioavailability variations, even in spite of their high membrane permeability [45, 57, 96, 121]. These characteristics impart difficulties for their penetration through the skin and present great challenges for the development of topical pharmaceutical systems [122, 123]. Therefore, various solubilization techniques have been employed to enhance drug solubilization such as the use of surfactants, water-soluble carriers, SEDDS, and solid dispersions [57, 124-128]. Several different solubilization methods have been used to increase the drug solubility and improve their bioavailability, thus improving their therapeutic activity [57, 96, 125, 127, 128, 136, 141, 144, 145].
Among these methods, self-emulsifying drug delivery system (SEDDS), a class of emulsion, has received special attention as a method to enhance oral bioavailability and dermal or transdermal delivery of poorly absorbed drugs thus reducing drug doses and providing more consistent absorption profiles [57, 96, 124-128, 136, 141, 144, 147-154]. Microemulsions are optically isotropic, thermodynamically and kinetically stable systems of water, oil, surfactant and co-surfactant, in addition to the drug of choice [25, 96, 136, 155]. They have been studied as drug delivery systems due to their ability to improve the solubility of poorly water-soluble drugs as well as enhancing their topical and systemic bioavailability [25, 96, 136, 155, 156]. Their high kinetic stability is attained only when the method of preparation , the components and the ratios used are appropriate [156, 157]. Nanoemulsions are emulsified systems with small droplet size (nanosized droplets) and high uniformity and homogeneity that provide several advantages such as solubilization and high stability [122, 135].
These components spontaneously emulsify when diluted with aqueous media, forming nanoemulsions under conditions of gentle agitation [57, 96, 134, 136, 137, 141, 153, 154, 158, 162-167]. The process of self-emulsification is highly dependent on the specific mixture of oils and surfactants used, their respective ratios and the temperature at the time of preparation [141, 181-183]. Thus, the choice of the most suitable surfactant is highly critical in determining long term stability of the preparation and possibly their interactions with biological systems [158, 184]. One of the main advantages of lipid formulations is keeping the drug in solution throughout the entire absorption process, since the hydrophobic core of the emulsion droplets can be used as a cargo container for drugs with poor water solubility [137, 158, 160, 161, 216].
The skin is the largest organ in the human body and it’s the main route for topical delivery of drugs [119]with absorption being dependent on several factors including, drug concentration, location of skin used, contact time, presence of hair and its amount, skin condition, type and properties of the used preparation and drug solubility [119, 262]. Dermal delivery is used to localize the treatment within the infected or damaged areas of the skin to improve the local effects and lower the systemic side effects [25, 119, 263, 264]. Topical application is a preferable route for local treatment because it is noninvasive, directly applied to the site of action and has markedly less systemic adverse effects [266, 267]. The efficacy of topically applied treatment in superficial mycoses depends on the type of lesion and the mechanism of action of the applied drug, as well as, the different properties of the formulation, such as viscosity, hydrophobicity and acidity [1, 25, 268]. Topical preparations intended for use on the skin have a wide variety of formulations The most common property of all of these preparations is their ability to adhere to the site of application for reasonable time before being worn off, removed or washed off [1, 119]. Currently, most topical formulations available in the market may cause systemic absorption or skin irritation and may not result in complete mycological cure [268, 273-276].
Fungal infections are one of the most prevalent public health issues in cutaneous infections worldwide and requires prolonged and costly treatment regimens with increasing cases [108, 122, 268, 278-280]. As a matter of fact, cutaneous mycotic infections especially dermatophytoses, tinea versicolor and those caused by Candida are the most common fungal infections [38, 122, 268, 282]. Superficial fungal infections can be classified as inflammatory or non-inflammatory [38, 267].
Tinea versicolor is more prevalent in tropical and subtropical areas where climates are hot with high humidity [2, 33, 38, 272]. It is among the most commonly prevalent superficial fungal skin infections affecting the stratum corneum in the world [38, 40, 91, 289-292]. It is typically caused by a lipophilic fungus of the Malassezia spp. called Malassezia furfur [64, 73, 291, 298, 310-315]. It is usually a highly recurring infection with a 65% relapse rate within the first year [38, 40, 91, 290, 298, 302-305] and up to 80 % in 2 years [4, 38, 40, 298, 306-309]. The disease is basically a pigmentation disorder with varying colors from white to brown depending on various factors [290, 298, 314, 324, 325]. Typically, it is clinically characterized by the presence of multiple well defined hypopigmented or hyperpigmented lesions [38, 40, 73, 91, 311, 313, 314, 327, 328]. Clinical diagnosis may be confirmed by examining the lesions under ultraviolet rays using a wood’s light, where they produce a yellow or gold fluorescence [38, 73, 298, 313, 314, 330]. It can also be confirmed by taking samples from the skin using a strip of sellotape or skin scrapings then microscopically examining these samples to detect the round spores and short curved hyphae of the fungus (spaghetti and meatball appearance) either alone or using parker blue ink or 10% – 20% potassium hydroxide as a stain or by cultures from scrapings of the lesions [38, 40, 73, 298, 313, 314, 330].
Different treatment strategies, either topical or systemic, and sometimes employing both types, using a wide variety of antifungal agents, can be used effectively for the treatment of tinea versicolor infections with high cure rates both clinically and mycologically [38, 40, 64, 73, 91, 290]. However, the efficacy of most of the currently available treatments is largely unpredictable with a wide range of side effects [298].
The present study aimed to design itraconazole or voriconazole-loaded SNEDDS. The resulting formulation could be used for both systemic, as well as, topical delivery. For the sake of this study, topical dermal delivery was used as an example for the different applications of the prepared gel.
5.2 Pre-clinical study
For itraconazole, the highest solubility in oils ranged from 12.812 ± 0.01 to 21.831 ± 0.01 mg/g and was recognized for cottonseed oil, oleic acid and lemon oil. Regarding surfactants or co-surfactants (according to relative HLB values), appropriate itraconazole solubility was detected for tween 40, tween 80, tween 85, PEG 200, PEG 600 and span 80 and ranged from 1.382 ± 0.03 to 4.44 ± 0.03 mg/g. Hence, these oils and surfactants were chosen for the phase study afterward.
For voriconazole, the highest oil solubility was measured in peppermint oil, oleic acid, lemon oil and cottonseed oil and ranged from 46.156 ± 0.01 to 404.245 ± 0.02 mg/g. For the surfactants/co-surfactants (depending on their relative HLB values), acceptable solubility values ranging from 53.113 ± 0.02 to 33.160 ± 0.01 mg/g were detected for tween 80, propylene glycol, PEG 200, PEG 400 and PEG 600. Thus, these oils and surfactants were the ones selected for the next step, the phase study.
The exact amount of the oil was accurately weighed into a screw capped tube followed by the addition of the surfactant and the co-surfactant to prepare the preconcentrate and then these preconcentrates were kept overnight and then diluted to form their corresponding nanoemulsion. The ternary phase diagrams of the different oil-surfactant-co-surfactant systems were plotted. A total of 54 different concentration ratios were prepared for each of the possible selected system.
from the resulting ternary phase diagrams select the system with the best possible properties that will help achieve the desired effect was selected. The selection was made based on:
• Presence of intermediate gel state.
• The highest drug solubility in the preconcentrate.
• The lowest amount of water needed to produce the lowest viscosity gel.
Based on the these criteria, for itraconazole, the preconcentrate consisting of 60% cottonseed oil and 40% span 80 resulted in development of the intermediate gel with the highest concentration of itraconazole (8 mg/g) in the prepared gel upon dilution of each 1 g of the preconcentrate with 0.7 ml of water (to produce the thinnest gel). Consequently, this system was selected for further studies.
For voriconazole, the preconcentrate consisting of 50% peppermint oil and 50% tween 80 resulted in development of the intermediate gel with the highest concentration of voriconazole (11.12 mg/g) in the prepared gel upon dilution of each 1 g of the preconcentrate with 0.8 ml of water (to produce the thinnest gel). Consequently, this system was selected for further studies.
The addition of either drug to their respective selected system did not have a noticeable effect on its phase behavior, nor did the addition of 5% w/w ethanol as a penetration enhancer.
Diluting 1 g of the preconcentrate of either selected system to 100 ml with water, phosphate buffer pH 5.4 or simulated sweat did not affect either the self-emulsification ability of the recommended systems, presence or absence of intermediate gel-like phase or the systems appearance. The nanoemulsions were formed immediately and were either clear (grade A) or slightly less clear (grade B) with a bluish or orange tinge.
The measured amounts of itraconazole and voriconazole in the freshly prepared samples were 13.599 mg (99.993%) and 19.9988 mg (99.994%) respectively, indicating that the entire added amount of the drugs (13.6 mg and 20 mg respectively) were solubilized with no precipitation.
For itraconazole, the mean globule size was 237.9 ± 46.11 or 236.9 ± 23.02 nm after dilution with distilled water or simulated sweat (containing 5% w/v SLS) respectively. There was no significant difference between the globule size in both media (P> 0.05), indicating that the formulation was not affected by the type of dilution media. Also, drug loading didn’t result in significant changes (p value > 0.05) in droplet size from the plain nanoemulsion, which exhibited a droplet size of 237.5 ± 33.04 nm. The corresponding polydispersity indices were 0.534 and 0.507 indicating a narrow globule size distribution, and reflecting the uniformity of the size distribution and particle diameter of the selected nanoemulsion. The measured zeta potentials were around -59.8 referring to high stability of the system.
For voriconazole, the mean globule size was 217.9 ± 31.17 or 214.4 ± 30.05 nm after dilution with distilled water or simulated sweat (containing 5% w/v SLS) respectively. There was no significant difference between the globule size in both media (P> 0.05), indicating that the formulation was not affected by the type of dilution media. In addition, drug loading didn’t result in significant changes (p value > 0.05) in droplet size from the plain nanoemulsion, which exhibited a droplet size of 215.5 ± 31.04. The corresponding polydispersity indices were 0.364 and 0.346 indicating narrow globule size distribution and reflecting uniformity in the size distribution and particle diameter of the selected nanoemulsion. The measured zeta potentials were around -35.7 referring to high stability of the system.
The viscosity of the intermediate gel was constant as the shear rate increased supporting Newtonian behavior of the gel [96, 122, 228]. The measured viscosity value was 1583.47 ± 36.2 cp for the itraconazole loaded intermediate gel and 1524 ± 42.1 cp for the voriconazole containing intermediate gel.
The pH of the selected nanoemulsion for itraconazole was 5.4, and for the selected voriconazole loaded nanoemulsion was 5.6. Both of which are acceptable for dermal applications.
Both, the selected SNEDDS and their respective nanoemulsions, either loaded with the drug or not, as well as the intermediate gels were stable and showed no visible changes in terms of appearance, clarity or phase separation over 12 months. At the end of the stability study, the drug content was unchanged (99.978% for itraconazole and 99.985% for voriconazole) which indicated high stability of the drug within the prepared formulation after storage for 12 months.
Both of the selected nanoemulsion intermediate gels had significantly higher antifungal activity (P value < 0.05) compared to that of their respective drug solutions against all tested species.
In the in-vitro release of itraconazole, the amount of itraconazole released after 1 hour was 34.52 % 5.26 and almost all of the drug content was released from the formulation by 4.5 hours (98.37 % 0.52). There was no lag time at the beginning of the experiment.
In the in-vitro release of voriconazole, the amount of voriconazole released after 1 hour was 32.82 % 1.07, while almost the entire drug content was released from the formulation by 4.5 hours (96.65 % 0.82). There was no lag time at the beginning of the experiment.
These results are very appropriate for a topical preparation that usually has an average skin contact time of about 6 h. This indicates that the entire dose is readily released from the preparation and available for absorption, allowing for good bioavailability.
The results of fitting the release data of both drugs to different kinetic models indicate that the release of both drugs from their respective selected nanoemulsion intermediate gels followed the Makoid-Banaker model with the highest R2 value (0.9963 for itraconazole and 0.9998 for voriconazole). It also had the lowest Akaike information criterion (AIC) of 57.9081 for itraconazole and 37.1625 for voriconazole. In addition to, the highest model selection criterion (MSC) at 4.9155 for itraconazole and 6.2406 for voriconazole.
Although both drugs were readily released from the preparation as per the results of the in-vitro release study, it was not as easily transmitted through the skin. The percentage of itraconazole that passed through the skin into the release media was 6 % 2.18 and 26.6 % 4.44 after 1 and 6 hours respectively against 34.52 % 5.26 and 99.98 % 0.33 in case of the in-vitro release study at the same time intervals. On the other hand, the percentage of voriconazole that passed through the skin into the release media was 5 % 1.24 and 25.11 % 6.27 after 1 and 6 hours respectively against 32.82 % 4.22 and 99.99 % 0.21 in case of the in-vitro release study at the same time intervals.
The results obtained from the ex-vivo skin permeation study suggested that the majority of the drug dose was retained inside the different layers of the skin instead of penetrating them and were supported by those obtained from measuring the amount of the drug retained in the layers of the used skin samples.
The amount of itraconazole in the homogenized skin samples was approximately 72.9 % of the total amount included in the selected preparation. Similarly, the amount of voriconazole in the homogenized skin samples was approximately 74.6 % of the total amount included in the selected preparation.
5.3 Clinical study
A double blind, placebo controlled randomized clinical study for the evaluation of the developed drug loaded self nanoemulsifying drug delivery system intermediate gel was conducted on 100 patients diagnosed with tinea versicolor. The patients’ age range was from 12 to 60 years. The patients were randomly selected from those attending the outpatient clinic of the dermatology department of the Minia university Hospital and divided into 5 groups with 20 patients in each group.
The patients were divided into 5 groups consisting of 20 patients each.
1- group 1 received the medicated formulation containing itraconazole once daily (ITZ once).
2- group 2 received the medicated formulation containing itraconazole twice daily (ITZ twice).
3- group 3 received the medicated formulation containing voriconazole once daily (VCZ once).
4- group 4 received the medicated formulation containing voriconazole twice daily (VCZ twice).
5- group 5 (the placebo group) received the unmedicated formulation twice daily (placebo).
Treatment was performed by applying the gel once or twice daily until full recovery was achieved with follow up once a week to reassess the condition. All adverse effects were checked during the study. The clinical improvement of the patients, treatment related side effects, patient and physician satisfaction and length of treatment were assessed during each follow-up visit. High resolution digital photographs were taken for lesions of patients using identical camera folder setting before starting treatment, on each follow up visit and after complete recovery.
There was a significant correlation between the patient’s sex and the type of infection with most of the female patients developing hyperpigmented lesions.
• For the itraconazole once daily application group
One patient (5%) showed excellent improvement after one week of treatment, while all other patient in the group (95%) showed good improvement after the first week. All patients (100%) were completely cured within two weeks of starting treatment with 2 patients (10%) cured in 10 days and one patient (5%) cured in 12 days. Four patients (20%) had some residual hypopigmentation which disappeared within one week. Physician satisfaction ranged from good (15%) to excellent (85%). Patients were somewhat satisfied (15%), satisfied (10%) or very satisfied (75%). None of the patients (0%) reported any treatment related side effects. No cases of recurrence were reported for at least 18 months post treatment.
• For the itraconazole twice daily application group
All patients (100%) were completely cured within one week of starting treatment with 2 patients (10%) cured in 3 days, one patient (5%) cured in 5 days and 2 patients (10%) cured in 6 days. One patient (5%) had some residual hypopigmentation which disappeared within one week. In all cases physician satisfaction levels ranged from good (5%) to excellent (95%). Patients were somewhat satisfied (5%), satisfied (5%) or very satisfied (90%). None of these patients (0%) reported any treatment related side effects. No cases of recurrence were reported for at least 18 months post treatment.
• For the voriconazole once daily application group
Five patients (25%) showed excellent improvement after one week of treatment, while all other patient in the group (75%) showed good improvement after the first week. All patients (100%) were completely cured within two weeks of starting treatment with 4 patients (20%) cured in 9 days, 2 patients (10%) cured in 12 days and 3 patients (15%) cured in 13 days. Two patients (10%) had some residual hypopigmentation which disappeared within. Physician satisfaction ranged from good (5%) to excellent (95%). Patients were satisfied (10%) or very satisfied (90%). None of these patients (0%) reported any treatment related side effects. No cases of recurrence were reported for at least 18 months post treatment.
• For the voriconazole twice daily application group
All patients (100%) were completely cured within one week of starting treatment with one patient (5%) cured in 4 days and 2 patients (10%) cured in 5 days. Two patients (10%) had some residual hypopigmentation which disappeared within one week. In all cases physician satisfaction levels ranged from good (5%) to excellent (95%). Patients were somewhat satisfied (5%) or very satisfied (95%). None of these patients (0%) reported any treatment related side effects. No cases of recurrence were reported for at least 18 months post treatment.