1Final year Student, Department of Pharmaceutics, Sri Vijay Vidyalaya College of Pharmacy, Dharmapuri, Affiliated to The Tamil Nadu Dr. M.G.R. Medical University Chennai -600 032, Tamil Nadu.
2Associate Professor, Department of Pharmaceutics, Sri Vijay Vidyalaya College of Pharmacy, Dharmapuri, Affiliated to The Tamil Nadu Dr. M.G.R. Medical University Chennai -600 032, Tamil Nadu.
3Principal, Department of Pharmaceutics, Sri Vijay Vidyalaya College of Pharmacy, Dharmapuri, Affiliated to The Tamil Nadu Dr. M.G.R. Medical University Chennai -600 032, Tamil Nadu.
Ketoconazole is a synthetic antifungal agent belonging to the group of triazole. Itis one of the commonly used antifungal agents for the treatment of local and systemic fungal infections. It is a BCS class II drug (low solubility and high permeability). On oral administration, its bioavailability is low due to poor aqueous solubility. The objective of this work is to prepare Ketoconazole nanoparticles and then incorporated into the freshly prepared gel for transdermal delivery, because patients with Skin infection such as athlete's foot, jock itch, ringworm, and certain kinds of dandruff. The dose is given at a higher level due to its low bioavailability. The nanoparticle transdermal approach was selected to provide prolonged release of Ketoconazole to the affected area that increase bioavailability, reduce the side effects, reduce large doses and increase the therapeutic efficacy METHOD AND RESULT Preformulation study was carried out which includes drug estimation method, drug excipients compatibility study and melting point determination. The drug estimation was found to be linear between 50 to 500µg/ml and the melting point was found to be 1460C. The FT-IR spectrum of drug-excipient was found to be satisfactory, which indicated that excipients were compatible. Ketoconazole nanoparticles were prepared by nanoprecipitation method with different ratio of drug to polymer (1:1, 1:2 and 1:3) and stabilizer (Poloxamer 188) (0.1%, 0.425% and 75%). Among the nine formulations F7 was selected as optimized formulation. The particle size, polydispersity index, Zeta found in the range of 48.5 to 83.6 nm, 0.127 to 0.244, -20.1 to -35.7 mv, 85.64% to 93.78% and 57% to 89.38%.. From the in-vitro drug release study, it was revealed that sustained release of same formulation last up to 12 hours. potential, % Entrapment efficiency and % drug content of all the formulations were found in the range of 48.5 to 83.6 nm, 0.127 to 0.244, -20.1 to -35.7 mv, 85.64% to 93.78% and 57% to 89.38%.. From the in-vitro drug release study, it was revealed that sustained release of same formulation last up to 12 hours.
Nanoparticles are one of the forms of novel drug delivery systems having the capability to release the drug at an optimum rate at the desired site of action. Nano particular formulations provide the liberty to use a wide range of polymers like synthetic, natural, biodegradable and non-biodegradable polymers.1 The size range of the nanoparticles is 1 to 1000nm, but for the purpose of drug delivery, nanoparticles in the range of 50 – 500 nm are acceptable depending on the route of administration Nanoparticles have become one of the most active areas of research in the field of drug delivery due to their ability to deliver the drugs to the right place, at appropriate times, and in the right dosage and in the right dosage. A wide sort of nanoparticles composed of a variety of materials including lipids, polymers and inorganic materials are developed leading to delivery systems that change in their physicochemical properties and their applications. The advantages of nano- encapsulation include the enhanced stability of labile drugs, controlled drug release and an enhanced drug bioavailability owing to the fact that particles in the nano-size range are efficient in crossing permeability barriers. Nanomedicine and nano delivery systems are a relatively new but rapidly developing science where materials in the nanoscale range are employed to serve as means of diagnostic tools or to deliver therapeutic agents to specific targeted sites in a controlled manner. Nanomaterials in improving both the efficacy of novel and old drugs (e.g., natural products) and selective diagnosis through disease marker molecules. Depending upon the tactic of preparation, nanoparticles, nanospheres or nano capsules are often obtained``
These have a monolithic type system in (matrix) in which drugs are either adsorbed or dispersed
The system in which the drug is confined to a cavity surrounded by a unique polymer membrane is known as nano capsules Method of preparation of nanoparticles: Several methods have been developed during the last two decades for the preparation of Polymeric nanoparticles. These techniques are classified as follows:
Nanoparticles obtained from dispersion of preformed polymer: Dispersion of drug in preformed polymers is a common technique used to prepare bio degradable nanoparticles from poly (lactic acid) (PLA), poly (D, L-glycolide) (PLG), poly (D, L-lactide-co-glycolide) (PLGA) and poly (cyanoacrylate) (PCA). These can be
accomplished by different methods described below.
EX: Solvent evaporation method: In this method, polymer solutions are prepared in volatile organic solvents (e.g. dichloromethane and chloroform) and emulsions are formulated by high-speed homogenization or ultrasonication and converted into a nanoparticle suspension on evaporation of the solvent for the polymer, which is allowed to diffuse through the continuous phase of the emulsion. In the conventional methods, two main strategies are being used for the formation of emulsions, the preparation of single-emulsions (e.g. oil-in water (o/w)) or double-emulsions, (e.g. (Water in-oil)-in-water (w/o)/w).
MATERIALS AND METHODS
MATERIALS:
The materials used in the present work are as follows:
Table no:1 List of Materials used and Manufacturers
EQUIPMENT:
The equipment used in the present work as a follow:
Table: 2 List of Instruments Used and Manufacturers
METHODS:
Preformulation studies:
Determination of ?max and Standard graph preparation:
Accurately weighed 50 mg of Ketoconazole is weighed and transferred into a 50ml volumetric flask, methanol was added to dissolve the drug, then the volume is made up to 50 ml, then from this solution 1 ml is taken and transferred into a 100 ml volumetric flask and made upto100 ml with PH 7.4 Phosphate buffer to get 10 ?g/ml solution. Then the prepared solution was scanned in the range of 200 to 400 nm by using pH 7.4 Phosphate buffer as a blank. The ?max was found to be 296 nm.
Preparation of Ketoconazole calibration curve:
50 mg of Ketoconazole was weighed accurately and carefully transferred in 100 ml volumetric flask and dissolved in methanol and the volume is made up to the mark with methanol (500µg/ml). From this solution aliquots of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5ml was pipetted out and diluted to 10ml to get 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500µg/ml by using pH 7.4 phosphate buffer. pH 7.4 phosphate buffer is used as a blank solution. Standard curve was prepared by plotting absorbance vs concentration at 296 nm using UV-Visible spectrophotometer.
Determination of melting point:
Determination of melting point gives an idea about purity of the drug. Melting point of Ketoconazole was determined by capillary method. Fine powder of Ketoconazole was filled in glass capillary tube (previously sealed on one end). The capillary tube is tied to assembly was kept on heating and temperature was allowed to increase gradually. Temperature at which the powder melts was noticed. thermometer and the thermometer was placed in tube containing liquid paraffin. The assembly was kept on heating and temperature was allowed to increase gradually. Temperature at which the powder melts was noticed.
Drug-excipients Compatibility study:
A successful formulation of a stable and effective solid dosage form depends on careful selection of the excipients that are added to facilitate administration, promote the consistent release and bioavailability of the drug and protect it from degradation. If the excipients are new and have not been used in formulations containing the active substance, the compatibility studies are of more importance.
FT-IR:
The compatibility of the drugs with the excipients was determined by subjecting the physical mixture of the drug and the polymers of the formulation to infrared absorption spectral analysis (FT-IR). Any change in the chemical composition of the drugs after combining it with the polymer was investigated with I.R spectral analysis.
Procedure:
The Ketoconazole nanoparticles were prepared by a nanoprecipitation method. The formulation plan is shown in. Ketoconazole 10mg and Eudragit L 100 10mg, 20mg and 30mg were dissolved in 3ml Ethanol. The internal organic phase solutions were slowly injected at the rate of (1ml/minute) into 20ml of the external aqueous solution containing stabilizing agent (Poloxamer 188) at various concentrations such as (0.1, 0.4 and 0.75% w/v) in double distilled water, and the mixtures were then stirred at 500 rpm for 4 hr at room temperature. Internal organic phase solutions are always composed of solvents, making the drug and Eudragit L 100 soluble completely, and the external aqueous phase comprises aqueous solution, sometimes with or without surfactant in it. The surfactant can penetrate into the Ketoconazole nanoparticles during the nanoprecipitation process to form a stable nanoparticle. The aqueous phase immediately turned into milky bluish opalescence due to the formation of the nanoparticle suspension. Ethanol was completely removed by rotary vacuum evaporation using a water bath maintained at 32°C. The Ketoconazole nanoparticles formed were isolated, washed three times with distilled water, and freeze-dried.
Table no 3 Formulation chart of Ketoconazole nanoparticles (F1- F9)
Preparation of nano particle-based gel:
Six formulations of Ketoconazole gel were prepared using carbopol 934 & carbopol 940 as a gelling agent with different ratios of 0.3%, 0.5% and 0.7 %. Specified quantity of carbopol 934 and carbopol 940 were soaked overnight as mentioned in the formulation chart shown in Table 4.5. Ketoconazole nanoparticle slurry was prepared by dissolving in a mixture of propylene glycol (penetration enhancer) & glycerine (moistening agent) under continuous stirring. To the carbopol slurry specified quantity of Ketoconazole nanoparticles slurry was slowly added with stirring. Propylene glycol (20 % w/w), Glycerine (10% w/v), Methyl paraben (0.03% w/w) & Propyl paraben (0.01 % w/w) were added slowly with continuous stirring until the homogenous gel was formed. The gel was neutralized with sufficient quantity of Triethanolamine and final volume was made to 50 ml with distilled water.
Table no 4 Formulation chart of Ketoconazole nanoparticles gel.
Evaluation of gel
Topical gel evaluated for following characters:
Measurement of pH:
The pH of gel formulations was determined by digital pH meter. 1g of gel is dissolved in 100 ml distilled water and stored for two hours. The measurement of pH of each Formulation is done in triplicate and average values are calculated.
Drug content studies:
Accurately weighed 1 g of gel was transferred into 10 ml volumetric flask containing 5 ml of saline phosphate buffer (pH 7.4) and stirred for 30 min followed by sonication. The volume was made up to 10 ml with saline phosphate buffer (pH 7.4). 5 ml of the above solution was further diluted to 10 ml with saline phosphate buffer (PH 7.4). The absorbance was measured using Shimadzu 1800 UV Visible spectrophotometer at 296 nm.
Viscosity measurement:
Viscosity of the gel was determined by using Brookfield viscometer. Accurately weighed 25gm of Ketoconazole gel was transferred to 50 ml glass beaker. Spindle no 6 was selected and it is immersed into the gel. The viscometer was operated at 10 rpm until the reading gets stabilized and reading was noted in centipoises. It was noted from the literature that the formulations after gelling should have a viscosity of 50 – 50,000 cps.
In-vitro diffusion studies:
In-vitro diffusion study was carried out in a Franz diffusion cell using cellophane membrane which is soaked overnight in distilled water. The membrane was tied to the donor compartment and mounted on the reservoir compartment of Franz diffusion cell containing 150 ml of pH 7.4 phosphate buffer. 1 gm of Ketoconazole gel was placed over the cellophane membrane of donor compartment. Whole set was placed on the magnetic stirrer. The study was carried out at 37±0. 5ºC and100 rpm for 12h. Samples were withdrawn from the sampling port of reservoir compartment at regular intervals and absorbance was measured using Shimadzu 1800 UV visible spectrophotometer at 296 nm.
Stability:
Stability testing of drug product is part of drug discovery and ends with the commercial product, to assess the drug and formulation stability, stability studies were done. The stability study was carried out for the optimized formulation (G5), subjecting to a temperature of 40 ± 2°C and 75 ± 5% RH and 4°C in refrigerator for 1 month. After 1 month the samples were analyzed for the physical characteristics, drug content and in-vitro diffusion study.
RESULT AND DISCUSSION:
Preformulation studies:
Determination of Melting Point:
The melting point of Ketoconazole was found to be 146 ?C.
Determination of wavelength maxima of Ketoconazole:
The solution was scanned in the range of 200-500 nm to fix the wavelength at which maximum absorption of Ketoconazole was observed. The ?max was found to be 296 nm in both methanol and pH 7.4 phosphate buffer.
Standard calibration Curve of Ketoconazole at ?max 296nm in phosphate buffer (pH 7.4): Ketoconazole obeyed Beer’s law in the range from 50-500 µg/ml. The absorbance is shown in the table and standard graph in figure.
Table no 5 Concentration and absorbance of the drug in prepared solutions:
Fig no 1 Standard calibration curve of Ketoconazole.
Drug-Excipient Compatibility Studies:
Fig. no 2 FTIR Characteristics Peaks of Pure Ketoconazole Drug
Table no 6 FT-IR Characteristics Peaks of Ketoconazole:
Fig no 3 FTIR Characteristics Peaks of Pure Ketoconazole Drug
Fig no 4 : FTIR Spectra of Ketoconazole and Eudragit L 100
Fig. no 5 FTIR Spectra of Ketoconazole and Polaxomer 188
Fig. no 6 : FTIR Spectra of Ketoconazole and Polaxomer 188
Fig. no 7 FTIR Spectra of Ketoconazole and Carbapol 940
Evaluation of nanoparticles:
Table no 7 Evaluation of nanoparticles by Optimized method (F1 to F9)
Table no 8 Evaluation of nanoparticles of Ketoconazole trial run
Fig no 8 Particle Size Distribution and Zeta potential of Formulation F7.
In-vitro diffusion study:
Table no 9 In-vitro diffusion release of Ketoconazole nanoparticle F1-F5
Table no 10 In-vitro diffusion release of Ketoconazole nanoparticle F6-F9
Fig no 9 In-vitro diffusion release of Ketoconazole nanoparticle (F1 to F5)
Fig no 10 In-vitro diffusion release of Ketoconazole nanoparticle (F6 to F9)
Stability studies:
Table no 11 Stability studies of Ketoconazole nanoparticles (F7)
Evaluation of Ketoconazole nanoparticle gel:
Table no 12 Evaluation of Ketoconazole nanoparticle gel
Table no 13 In-vitro diffusion release of Ketoconazole nanoparticle gel (G5)
Fig no 11 In-vitro diffusion release of Ketoconazole nanoparticle gel (G1 to G6)
Drug release kinetics of formulation G5
Table no 14 Kinetics of drug release of G5 Formulation
Fig no 12 Zero order plot for drug release kinetics of G5 formulation.
Fig. 13 First order plot for drug release kinetics of G5 formulation.
Fig no 14 Higuchi plot for drug release kinetics of G5 formulation.
Fig no 15 Korsmeyer Peppa’s plot for drug release kinetics of G5 formulation
Stability studies:
Table no 15 stability studies of Ketoconazole nanoparticle gel (G5)
CONCLUSION
REFERENCE
Bhakti B. Bansod, Rekha Goukonde, Gajanan Sanap, A Review On Acacia Arabica And It's Medicinal Uses, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 5, 1-6. https://doi.org/10.5281/zenodo.11208130
10.5281/zenodo.11208130
