5.4.1.1. Screening of drugs:
Various antiviral drugs like abacair sulphate, zidoudine, tenofoir, efairenz, lamividine, and staudine were selected. The comparitive table of selected drugs was prepared to find out the suitability of drugs for proniosome formulation.Based on the solubility, dosage frequency, half life, suitability to controlled release and adverse drug reaction abacavir sulphate was screened.
5.4.1.2. Preparation of reagents:
5.4.1.2.1. Preparation of phosphate buffer pH 7.4:
Dissolve 6.8 gm of potassium dihydrogen orthophosphate and 1.56 gm of sodium hydroxide in 900 ml of water, adjust the pH 7.4 with sodium hydroxide solution and dilute the water to produce 1000 ml.166
5.4.1.2.2. Preparation of phosphate buffer pH 6.8:
Dissolve 28.8 gm of disodium hydrogen phosphate and 11.45 gm of potassium dihydrogen phosphate in sufficient water to produce 1000 ml.166
5.4.1.2.3. Preparation of 0.1N HCl:
Measure 8.5 ml of hydrochloric acid in 1 litre standard volumetric flask and make up the volume using demineralized water.
5.4.1.3. Calibration of standard curve:
Preparation of stock solution:
Accurately weighed abacavir sulphate which is equivalent to 100 mg of abacavir was taken in three 100 ml standard volumetric flask with proper label. The content of first, second and third flask were dissolved in pH 7.4 phosphate buffer, pH 6.8 phosphate buffer and 0.1N hydrochloric acid respectively. The volume of each flask was made upto 100 ml using respective buffer solutions to obtain a stock solution-1 containing 1000µg/ml abacavir.
Identification of analytical wave length:
The three stock solutions were appropriately diluted with their respective buffer solutions and scanned using the UV visible spectrophotometer (UV-260, Shimadzu, Japan) in wavelength range in between 400 nm to 200 nm.
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Standard graph:
From the stock solution-1, of three standard flask 10 ml of the contents were pipetted out into three 100ml standard volumetric flask and made upto the marks using respective buffer solutions (stock solution2). From thesethree stock solutions -2, aliquots of 2ml, 4ml, 6ml, 8ml and 10ml were pipetted out into a series of 100 ml standard volumetric flasks and the volume was made upto the mark with respective buffer to get drug concentration in the range of 2 to 10 µg/ml. The absorbance of the resulting solutions were then measured at 285 nmincase of pH 7.4 and pH6.8 phophate solution and 295 in case of 0.1N hydrochloric acid using UV double beam spectrophotometer against proper buffer solutions as blank. Three standard curves were obtained by plotting concentration (µg/ml) values in X-axis and absorbance values in Y-axis.
5.4.1.4. Organoleptic properties of abacavir sulphate:
Organoleptic properties like color, odor and taste of abacavir sulphate were recorded.188
5.4.1.5. Particle size analysis:
Particle size distribution of the abacair sulphate was estimated by analytical sieving method. The sieves were stacked on top of one another in ascending degrees of coarseness. The abacair sulphate powder was placed on the top sieve. The nest of sieves was subjected to a standard period of agitation. The weight of material retained on each sieve was exactly determined.189
5.4.1.6. Solubility Studies of pure drug:
An excess amount of abacavir sulphate was added to each of distilled water, chloroform, ethanol, nbutanol,methanol, 0.1 N hydrochloric acid and phosphate buffer (pH 6.8 and 7.4). The mixtures were then kept in a well closed air tight containers at ambient temperature for 24 hours in vortex mixer to get equilibrium. The equilibrated samples were centrifuged at 3000rpm for 5 minutes. Aliquot portions of the supernatants were taken and properly diluted with phosphate buffer (pH 7.4) for quantification of abacavir sulphate spectrophotometrically at 285 nm.56,85,132
Approximate solubility of drug was indicated from the following limits.166
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Very soluble: 1 part of the substance is soluble in less than 1 part of the solvent.
Freely soluble: 1 part of substance is soluble in 1 to 10 parts of solvent.
Soluble: 1 part of substance is soluble in 10 to 30 parts of solvent.
Sparingly soluble: 1 part of substance is soluble in 30-100 parts of solvent.
Slightly soluble: 1 part of substance is soluble in 100 to 1,000 parts of solvent.
Very slightly soluble: 1 part of substance is soluble in 1,000 to 10,000 parts of solvent.
Practically insoluble or insoluble: More than 10,000 parts of solvent is required to dissolve 1 part of substance.
5.4.1.7. Partition co-efficient of pure drug:
Accurately weighed quantity of abacavir sulphate equivalent to 100mg abacavir was dissolved in pH 7.4 phosphate buffer and is shaken with theother partitioning organic solvent (n-butanol) for 30minutes, allowed tostand for 5 minutes, and then removed the lower and upper parts separately. Then the partitioning coefficient was obtained using the formula as follows.
Kwo= Concentration of drug in organic phase/Concentration of drug in aqueous phase56
5.4.1.8. IR spectrum of pure Abacavir sulphate:
The drug abacavir sulphate was confirmed by comparing the IR spectrum of the drug with published IR spectrum of abacavir sulphate in Indian Pharmacopoeia.161 5.4.1.9. Drug – excipients compatability studies by FT-IR spectroscopy:
The Drug – excipients compatibility studies were performed in order to confirm absence of any interaction between drug and excipients.145 Disappearance of an absorption peak or reduction of the peak intensity combined with the appearance of new peaks give a clear evidence for interactions between drug and excipient.129
Compatibility studies were performed by preparing blend of different excipient with drug. These blend were placed in air tight screw cap amber colored
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vials at accelerated condition like 40°C/75% RH for one week and carry out FT-IR analysis with saturated potassium bromide using pellet making method. Abacavir sulphate was also stored alone as a reference and each drug - excipient blend is further evaluated for changes like caking, liquefaction, discoloration and odor (or) gas formation. 56,190
Table 5.4.1: Drug :excipient compatibility protocol
Sl.No Composition Quantity Ratio
(Drug:Excipient)
1 Abacavir Sulphate 100mg -
2 Abacavir Sulphate + Cholesterol 150 mg 1:0.5
3 Abacavir Sulphate+ Maltodextrin 300 mg 1:2
4 Abacavir Sulphate+ Span 20 150 mg 1:0.5
5 Abacavir Sulphate + Span40 150 mg 1:0.5
6 Abacavir Sulphate + Span60 150 mg 1:0.5
7 Abacavir Sulphate + Span 80 150 mg 1:0.5
8 Abacavir Sulphate + Tween 20 150 mg 1:0.5
9 Abacavir Sulphate + Tween 40 150 mg 1:0.5
10 Abacavir Sulphate + Tween 60 150 mg 1:0.5
11 Abacavir Sulphate + Tween 80 150 mg 1:0.5
12 Abacavir Sulphate + Dicetyl
Phosphate 125 mg 1:0.25
5.4.1.10. Determination of abacavir sulphate amount to be used in a proniosome formulation:
Molecular weight of abacavir is 286.33232 and molecular weight of abacavir Sulphate is 335.37132. The total amount of abacavir sulphate to be used in formulation to fulfill the required abacavir was calculated.
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5.4.2. Formulation:
5.4.2.1. Concentration and process optimization:
The concentrations of nonionic surfactant and cholesterol were optimized to develop the stable dosage form with different nonionicsurfactants (spans and tweens) at 100, 150, 200 and 250 μM concentrations with an equalratio of cholesterol.
Although vesicles were formed in all concentrations, the encapsulation efficiency was found to be very less except at 250 μM ratio. Thus 250:250μM ratio of surfactant:cholesterol was preferred for present formulation. The process-related variables like speed of rotation of flask, hydration medium and hydration time were optimized by trial and error method.
5.4.2.2. Abacavir sulphate niosome preparation:
Niosome containing abacavir sulphate formulations were prepared by thin film hydrationmethod. The surfactants, cholesterol and dicetyl phosphate in 250: 250:
5µM ratios were accurately weighed and transferred into a long necked 100 ml round-bottom flask and dissolved in 10 ml chloroform. The flask was attached to a rotary evaporator and the organic solvent wasslowly evaporated at 60°C under reduced pressure at 100-150 rpm such that a thin dry film of the constituents was formed on the inner wall of the flask. Any excess chloroform was removed by leaving the flask in a desiccator under vacuum overnight. The dried thin film was then hydrated with 10 ml pH 7.4 phosphate buffered saline containing 25 mg abacavir sulphate, (or 10 ml pH 7.4 phosphate buffered saline) by rotating the flask in the same rotary evaporator under normal pressure at 60°C in order to ensure complete hydration of the film. The prepared niosomal preparations (drug loaded/ blank) were stored in a refrigerater for the further evaluations. 191,192
Table 5.4.2: Composition of abacavir sulphate niosomal formulations without charge inducer
S.No Formulation