| Abstract: |
Ketoconazole is a broad-spectrum antimycotic agent that can be used orally or topically in the treatment of candidiasis and other fungal infections. Ketoconazole is practically insoluble in water that limits its practical use, especially for the topical treatment of coetaneous diseases.In the present thesis various techniques were applied to improve the solubility, antimycotic activity and dissolution rate of ketoconazole, viz.: complexation with certain cyclodextrins (CDs) and solid dispersion with certain macromolecules. Furthermore, the physicochemical characteristics of the prepared solid systems were studied by IR-spectroscopy, DSC and powder X-ray diffraction. The release of ketoconazole from those systems was also investigated. Additionally, ketoconazole was formulated into vaginal pessaries and their clinical efficiency was evaluated.As an integral part of this thesis, attempts were made to discuss the main mechanism(s) responsible for dissolution rate improvement of the drug and to shed some lights on the mechanism of interaction of the drug with certain cyclodextrins and macromolecules in liquid and solid states.Accordingly, this thesis comprises the following parts:Part I: Investigation of Cyclodextrin Inclusion Complexes and Macromolecule Interactions toward Ketoconazole.The work in this part was divided into three chapters.Chapter 1Effect of certain cyclodextrins on the solubilziation, antimycotic activity and dissolution rate of ketoconazole.The aim of the study in this chapter was to investigate the effect of certain natural CDs and CD derivatives namely; -CD, -CD, hydroxypropyl--CD (HP--CD) and dimethyl--CD (DM--CD) on the solubility and dissolution rate of ketoconazole. In addition, the antimycotic activity of the soluble inclusion complexes of ketoconazole with the studied CDs was evaluated.To fulfill this goal, the work in this chapter includes the following:A- Effect of pH on ketoconazole solubility in absence and in presence of different CDs.The data from this study revealed that:1- The solubility of ketoconazole increases in the studied pH range in presence of different CDs.2- The solubility of ketoconazole increases as pH value decreases in the studied pH range in absence and in presence of the investigated CDs.B- Solubility study of ketoconazole with the investigated CDs:Solubility study of ketoconazole with different concentrations of CDs in phosphate buffer of pH 5 at 30°C was done. Moreover, the effect of various temperatures on the solubility of the drug with -CD was evaluated in the same buffer solution.The results obtained from this study revealed that:1- The solubility of ketoconazole is improved in phosphate buffer of pH 5 containing various concentrations of the studied CDs, showing AL-type phase solubility diagrams.2- DM--CD has the heighest solubilizing capacity toward ketoconazole among the studied CDs and the complex formed was found to have the heighest stability constant.3- The solubility of the drug in -CD is improved by increasing the temperature from 30 to 45°C.C- Determination of the drug partition coefficient:Ketoconazole partition coefficient between n-octanol and phosphate buffer of pH 5 in absence and in presence of different concentrations of -CD was determined.The data from this study showed that: The partition coefficient of ketoconazole is more than 1, whereas its value decreases in presence of -CD due to the hyDROPhilic nature of the inclusion complex as compared to the drug alone.D- The antimycotic activity of ketoconazole:The antimycotic activity of the soluble inclusion complexes between ketoconazole and the investigated CDs was measured by agar-cup diffusion method.The results obtained from this study revealed that:1- The inhibition zone of candida albicans increases as the concentration of CD increases.2- Inclusion complex of ketoconazole with DM--CD shows the heighest antimycotic activity among the studied CDs.E- Interaction of ketoconazole with CDs in the solid state:This was studied by the following:1- Preparation of solid inclusion complexes:The solid complexes of ketoconazole with the investigated CDs were prepared by coevaporation and cogrinding methods.2- Characterization of the prepared solid complexes:The physicochemical properties of the solid inclusion complexes of ketoconazole with CDs were investigated by IR-spectroscopy, powder X-ray diffractometry and DSC.The obtained results revealed the formation of amorphous inclusion complexes of the drug with both HP--CD and DM--CD by cogrinding method.3- Dissolution study of ketoconazole from the prepared systems with CDs:The dissolution rate of ketoconazole from the prepared systems with the studied CDs was measured according to the dispersed amount method.The results obtained from the dissolution studied indicated that:1- The dissolution rate of ketoconazole from the prepared systems with CDs is improved in the following order: coground mixture > coevaporate > physical mixture > drug alone.2- The enhancement of dissolution rate of the drug is confirmed by the marked DROP in t50% and the increase in the relative dissolution rate (RDR) value of the drug.3- The increase in solubility, decrease in crystallinity, as well as inclusion complex formation are the cause of the enhancement of the dissolution rate of ketoconazole.4- The effect of various CDs on dissolution rate of ketoconazole can be arranged in the following order: DM--CD > -CD > HP--CD > -CD.Chapter 2Effect of certain macromolecules on the solubilization and dissolution rate of ketoconazoleThe aim of this chapter was to study the interaction of ketoconazole with some macromolecules in solution and in solid states. Two types of macromolecules were used throughout this work, namely; PEGs and PVPs in various weight ratios.The work in this chapter includes the following:A – Solubility study:Solubility of ketoconazole in the presence of PEGs and PVPs at different temperatures (30, 37 and 45°C) as carried out in phosphate buffer of pH 5.The results of this study showed the following:1- The solubility of ketoconazole is improved by increasing the concentration of either PVPs or PEGs in phosphate buffer of pH 5.2- The solubilizing efficiency of PVPs toward ketoconazole is higher than that of PEGs.3- The solubility of ketoconazole is improved by rising the temperature from 30 to 45°C in presence of either PVPs or PEGs.B- Preparation of solid dispersions of ketoconazole with macromolecules:The solid dispersions of ketoconazole with PEG 4000 and PEG 6000 were prepared by coevaporation and cofusion method in 4:6, 3:7, 2:8 and 1:9 (drug: carrier) weight ratios.Furthermore, the solid dispersions of ketoconazole with PVP 40000 were prepared by coevaporation and cogrinding methods in weight ratios 1:3, 1:5, 1:7 and 1:9 (drug: carrier).C- Characterization of the prepared solid dispersions:IR-spectroscopy, powder X-ray diffractometry and DSC studies were employed for characterization of the prepared systems.from the obtained results, it could be concluded that:1- IR spectra of the solid dispersions indicated that ketoconazole is compatible with the studied macromolecules as there is no interaction between ketoconazole and these carriers.2- Phase diagrams of ketoconazole with either PEG 4000 or PEG 6000 constructed using DSC data gives monotectic phase type.3- Solid dispersions of ketoconazole and PEGs prepared by coevaporation and comelt methods show exactly the same thermal behavior and similar diffraction pattern as that of the physical mixtures of the same composition.4- DSC and powder X-ray diffraction data show the formation of amorphous solid dispersion of ketoconazole with PVP 40000 by cogrinding method.D- Dissolution study of ketoconazole from the prepared solid dispersions:The dissolution rate was measured according to the dispersed amount method.The results obtained from dissolution study indicated that:1- There is a marked increase in the dissolution rate of ketoconazole from the prepared solid dispersions with either PEGs or PVP when compared to that of the corresponding physical mixtures and the drug alone.2- The dissolution rate of ketoconazole from its solid dispersions with macromolecules is dependent on the molecular weight of the carrier, the method of preparation and the carrier weight ratio in the system.3- The comelt of the drug with either PEG 4000 or PEG 6000 gives the heighest dissolution rate among the studied systems.4- The dissolution rate of ketoconazole reaches 100% from its coground mixture with PVP 40000 in weight ratio 1:9.Chapter 3Effect of polyethylene glycol, polyvinyl pyrrolidone and boric acid on ketoconazole--cyclodextrin binary systemThe aim of the study in this chapter was to investigate the combined effect of -CD and boric acid or PEG 4000 or PVP 40000 on ketoconazole solubility. Moreover, the influence of these substances on the physico chemical properties of ketoconazole -CD binary system was evaluated.To fulfil this goal, the work in the chapter includes the following:A- Solubility study:Solubility study was carried out to investigate the effect of addition of 1% (w/v) of either boric acid or PEG 4000 or PVP 40000 on complexation ability of -CD toward ketoconazole in phosphate buffer of pH 5 at 30°C.The results attained are as follows:1- AL-phase solubility diagrams are obtained for ketoconazole with -CD in the studied ternary systems.2- Boric acid, PEG 4000 and PVP 40000 show increase in ketoconazole solubilization by -CD.3- The value of stability constant of ketoconazole with -CD was increased upon the addition of 1% (w/v) of either boric acid, PEG 4000 or PVP 40000.4- Ketoconazole/-CD/PVP 40000 is the most effective ternary system for improving ketoconazole solubility.B- Preparation of solid ternary systems:The ternary systems of ketoconazole/-CD/boric acid (1:1:1 M) were prepared by coevaporation and cogrinding methods.In addition, the ternary systems of ketoconazole/-CD (1:1 M) in presence of 9% of either PEG 4000 or PVP 40000 were prepared by coevaporation and cogrinding methods.C- Characterization of the prepared ternary systems:Powder X-ray diffractometry and DSC studies were employed for characterization of the prepared ternary systems.Powder X-ray and DSC data revealed the formation of amorphous inclusion complex of the drug with -CD in case of all the studied ternary systems prepared by grinding methods.D- Dissolution study of ketoconazole from the various ternary systems:The dissolution rate of ketoconazole from the prepared ternary systems was measured according to the dispersed amount method.The results obtained from dissolution studies indicated that:1- A marked increase in dissolution rate of ketoconazole is obtained in case of coground mixtures and coevaporates of ternary systems than that of the corresponding physical mixtures and the plain drug.2- The increase of ketoconazole dissolution rate is higher from the ternary systems than from the respective binary ones.3- The heighest drug dissolution rate is obtained from ketoconazole/-CD/PVP 40000 ternary system prepared by co-grinding method.4- The addition of small amounts of either boric acid, PEG 4000 or PVP 40000 to ketoconazole/-CD complex is a useful strategy for improving the solubilizing and complexing ability of -CD toward the drug. Thus, allowing less amount of -CD to be used to solubilize a given amount of the drug offering an evident economic advantage as well.Part II: Formulation and Clinical Evaluation of Ketoconazole Vaginal pessaries:The work in this part was divided into two chapters.Chapter 1Formulation and in vitro evaluation of ketoconazole vaginal pessariesThe aim of the work in this chapter was to formulate ketoconazole into vaginal pessaries using both water-soluble and fatty suppository bases.The fatty bases used were Cocoa butter, Witepsol E75, Suppocire AM and Suppocire CM. The water-soluble bases used were PEG 1500 alone and a blend of polyethylene glycols (PEG 1500: PEG 400 in weight ratio of 90:10 and PG 4000: PEG 1000 in weight ratio of 75:25).The influence of the complexation of ketoconazole with -CD in both binary and ternary systems on the physical characters and drug release from the prepared vaginal pessaries was studied. All the vaginal pessaries were prepared by the fusion method.All the formulations were evaluated with regard to the drug content, uniformity of weight, disintegration time, melting range and hardness test. The prepared vaginal pessaries of ketoconazole were found to be acceptable with regards to all the tested properties.Release studies of ketoconazole from the prepared vaginal pessaries were carried out by using the USP XXV dissolution apparatus II (Paddle method) in phosphate buffer of pH 5 at 37°C.The obtained results revealed that:1- The release rate of ketoconazole is higher from vaginal pessaries prepared with water-soluble bases than from fatty-based vaginal pessaries.A total of 242 samples from Cystic fibrosis (CF) patients, septic wound, urinary tract (UT) infection, otitis media and environment, 31 Burkholderia cepacia isolates were collected from pathogenic clinical and environmental samples represent 12.8%. Samples of cystic fibrosis of respiratory tract (175), septic wounds (42), urinary tract (2) and otitis media (2) and non pathogenic environmental samples (21) from soil, macerated onions and water canals. B. cepacia isolated from CF patients represents 9.2%, while it represents 19% from septic wound samples. Only one B. cepacia isolate was characterized form UT and non from otitis media. In addition 28.6% were discriminated from environmental samples.The B. cepacia isolates were identified by cultural, morphological and biochemical characteristics after isolation on cetrimide-polymexin agar media and selective Mast media for B. cepacia.The chromosomal DNA of the selected isolates was extracted enzymatically using lysozyme and proteinase K enzymes and analyzed by electrophoresis on 1% agarose gel. Out of 31 isolates, 21 chromosomal DNA of selected B. cepacia isolates (7 CF, 1 UT, 8 wound and 5 environmental) were analyzed and recognized by PCR using species-specific primers for 35 cycles. The B. cepacia complex rec A gene (1,040bp) is amplified using primers BCR1 (TGA CCG CCG AGA AGA GCA A) and BCR2 (CTC TTC TTC GTC CAT CGC CTC), which targets the 5’ and 3’ ends of the rec A gene locus, respectively. After PCR and gel electrophoresis, a significant DNA band of about 1024 bp was detected with chromosomal DNA template of B. cepacia isolates
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