Azithromycin is a semi-synthetic macrolide antibiotic drug, effective against a wide variety of bacteria. It is primarily used to treat the bacterial infections associated with weaker immune system. Prednisolone is a synthetic corticosteroid, used for suppressing the immune system and inflammation. When used in combination, both the drugs are very much effective in the management of inflammatory conditions or diseases in which the immune system plays an important role. The aim is to study the preformulation parameters for niosomal gel for topical use. The objective of Preformulation study is to generic information useful to the formulator in developing stable and bioavailable dosage form. The use of Preformulation parameter maximize the chances of getting a formulation which is safe, efficacious and stable product and at the same time provide optimization of the drug product quality. Administration of conventional tablets of prednisolone has been reported to exhibit delayed release and unwanted side effects so prednisolone loaded niosomes were developed and azithromycin which tend to cause allergic reaction was incorporated into gel base provide rapid penetration through skin , improve therapeutic performance, restrict action to the target cell and improve patient compliance, hence the objective of the study was made to develop sustained release gel containing azithromycin and niosomal vesicles of prednisolone using Carbopol as a polymer which will controlled the release of drug, increasing the bioavailability of the drug and thus decreasing the dosing frequency of the drug. The Preformulation studies were carried out for identification (physical appearance, melting point, and uv spectrophotometer), solubility profile, TLC, FTIR, compatibility studies, simultaneous estimation. All the observation and results showed that the azithromycin and prednisolone serve as suitable candidate for Topical drug delivery system that may improve the bioavailability.
Keywords: Niosome; Topical gel; Preformulation; Compatibility; Simultaneous estimation; , sustain
Topical niosomal gel has been in use for dermatological disease since very long © time. Azithromycin (Figure 1) is a semieffective against a wide variety of bacteria. It is primarily used to treat the bacterial infections associated with weaker immune system. Prednisolone (Figure 2) is a synthetic corticosteroid, used for suppressing the immune system and inflammation. When used in combination, both the drugs are very much effective in the management of inflammatory conditions or diseases in which the immune system plays an important role [1]. The objective of Preformulation study is to generic information useful to the formulator in developing stable and bioavailable dosage form. The use of Preformulation parameter maximize the chances in formulation an safe, efficacious and stable product and compatible, at the same time provides simultaneous estimation studies and product quality. The Preformulation studies were carried out for identification (physical appearance, solubility studies, melting point, and uv spectrophotometer, IR spectra TLC, estimation of drugs). Niosomal gel has been explored extensively for Topical application to enhance skin penetration as well as skin retention of the drug. It provides effective and immediate release of the drugs [2]. Prednisolone loaded niosomes were used to provide sustained release and protect the drug from external environment, which further are being evaluated before
Solubility study of azithromycin was performed in 0.1 N HCl & water. Excess of AZI was dissolved separately in the above 2 solvents and shaken continuously for 24 hours in the mechanical shaker at 25±2°C. Solutions were filtered and absorbance was recorded using UV spectrometer (Systronic 2230) and the amount of AZI dissolved in 10 ml of 0.1N HCl & water was calculated [5].
The melting point of the drug was performed by capillary method. In this, drug was filled in the capillary tube sealed at one end to a height of 3 mm from closed end and capillary was introduced into digital melting point apparatus (SSS Pvt. Ltd.). The temperature range at which drug melt was noted down.
Characterization of AZI was done by FTIR spectroscopy. The drug was mixed with KBr & pressed into a very thin pellet which was then observed under IR spectrophotometer and the spectrum obtained was interpreted [6].
313
AZI was dissolved in acetone, applied on TLC plate & run in the saturated chamber containing mobile phase. Mobile phase was prepared by taking mixture of ethylacetate and hexane in the ratio 1:1(fried et al; 1999). Rf value was then calculated.
(λ ) of azithromycin
For the standardization of the drug by using UV spectroscopy, the drug is firstly subjected to wavelength scan for determination of absorbance maxima (λ ). A stock solution (1000μg/ml) of max drug was prepared by dissolving 100 mg drug in 20 ml, 0.1N HCl in 100 ml volumetric flask and volume was made up to 100 ml with respective dissolution media (phosphate buffer saline pH 6.5). The samples were scanned between range of 200-400 nm by using UV-visible spectrophotometer. The wavelength at which maximum absorbance observed was selected as the analytical wavelength of the drug for that particular buffer media.
The Calibration curve of the drug was plotted in phosphate buffer saline (pH 6.5) using 0.1N HCl as a cosolvent to dissolve the drug [7]. A stock solution of drug was prepared and was serially diluted with phosphate buffer saline pH 6.5 to obtain the concentration range of 100-800μg/ml respectively. The dilutions were analyzed spectrophotometrically at λmax of 298 nm using phosphate buffer saline (pH 6.5) as a blank.
Solubility study of Prednisolone was performed in methanol & water. Excess of drug was dissolved separately in the above 2 solvents and shaken continuously for 24 hours in the mechanical shaker at 25±2°C. Solutions were filtered and absorbance was recorded using UV spectrometer (Systronic 2203) and the amount of Prednisolone dissolved in 10 ml of metanol & water was calculated.
The melting point of the drug was determined by capillary method. In this drug the was filled in the capillary tube sealed at one end to a height of 3 mm from closed end and capillary was introduced into digital melting point apparatus. The temperature range at which drug melts was noted down.
Characterization of Prednisolone was done by FTIR spectroscopy. The drug was mixed with KBr & pressed into a very thin pellet which was then observed under IR spectrophotometer (FTIR 8400 S, Shimadzu) and the spectrum obtained was interpreted.
Prednisolone was dissolved in acetone, applied on TLC plate & run in the saturated chamber containing mobile phase. Mobile phase was prepared by taking mixture of ethyl-acetate and hexane in the ratio 1:1(fried et al; 1999). Rf value was then calculated.
For the standardization of the drug using UV spectroscopy, the drug is firstly subjected to wavelength scan for 314 determination of absorbance maxima (λ ) [8]. A stock solution (1000μg/ml) of max drug was prepared by dissolving 100 mg drug in 20 ml of methanol in 100 ml volumetric flask and volume was made up to 100 ml with respective dissolution media (phosphate buffer saline pH 6.5). The samples were scanned over the range of 200-400 nm by using UV-visible spectrophotometer. The wavelength at which maximum absorbance observed was selected as the analytical wavelength of the drug for that particular buffer media.
The Calibration curve of the drug was plotted in phosphate buffer saline pH 6.5 using methanol as a cosolvent to dissolve the drug. A stock solution of drug was prepared and was serially diluted with phosphate buffer saline pH 6.5 to obtain the concentration of 10-50μg/ml respectively. The dilutions were analyzed spectrophotometrically at λmax of 245 nm using methanol and phosphate buffer saline pH 6.5 as a blank [9].
The specified amount of drugs and the excipients (Span 60, cholesterol, carbopol, triethanolamine, methyl paraben, propylene glycol, glycerin) were weighed separately, mixed in ratio of 1:1 and filled in seprate vials. The vials were then stored under 2 different conditions at 2- 8°C & 40° C with 75% RH. Observations of th th all mixture were done on 0 day, 15 day th & 30 day. The compatibility of drugs with oily bases (Span 60, cholesterol, carbopol, triethanolamine, methyl paraben, propylene glycol, glycerin) was studied by thin layer chromatography [10].
Accurately weighed 10 mg of AZI was transferred to 10 ml volumetric flask and volume was made up with 0.1 N HCl (used as cosolvent as AZI is not completely soluble in phosphate buffer saline (pH 6.5) to get a solution of concentration 1000μg/ml. 1 ml of stock solution was diluted with phosphate buffer saline (pH 6.5) upto 10 ml to get a concentration of 100μg/ml and then further dilutions were made to obtain the concentration range of 10-45μg/ml using phosphate buffer saline (pH 6.5). Solution of PRE was prepared in methanol (used as cosolvent) in a similar way to obtain the concentration range of 10-90μg/ml by dilution with phosphate buffer saline (pH 6.5). Both the solutions were scanned in the spectrum mode over the range 200-400nm [11].
Concentration of each component was determined by using simultaneous estimation equation A = E C + E C 298 nm 1 1a 1 2a 2………………….
A2 = E1b C1 + E2bC2…………………245 nm A1 =absorbance value of sample at 298 nm A2 =absorbance value of sample at 245 nm st E = absopitivity of 1 drug at 298 nm 1a st E1b = absopitivity of 1 drug at 245 nm nd E2a = absopitivity of 2 drug at 298 nm nd E = absopitivity of 2 drug at 245 nm 2b st C1 = concentration of 1 drug in μg/ml nd C = concentration of 2 drug in μg/ml 2 The absorbance of sample solutions of AZI and PRE were measured at 298 nm and 245 nm respectively. The results were calculated by the following formula using Vierodt’s method. [15] A1 = ax1 Cx + ax2 Cy at 298 nm A2 = ay1 Cx + ay2 Cy at 245 nm Where, A1 and A2 are absorbance of diluted mixture of drugs at 298 nm and 245 nm 315 respectively, Cx and Cy are the concentration of AZI and PRE respectively (μg/ml), ax1 and ax2 are absorptivities of AZI at 298 nm and 245 nm respectively, ay1 and ay2 are absorptivities of PRE at 298 nm and 245 nm respectively [12].
Azithromycin was found to be Insoluble in water, ethanol, phosphate buffer saline, sparingly soluble in methanol, soluble in 0.1N HCl (Table 1). Prednisolone was very slightly soluble in water, soluble in methanol, dioxane. (Table 2).
Observed melting point (Table 3) of azithromycin and prednisolone was found similar to reported one which shows drugs are pure and stable.
IR spectra of azithromycin and prednisolone was obtained and interpreted by identifying the the value of characteristics (Figure 3 & Figure 4) -1 showed sharp peak at 1650-1850 cm corresponding to stretching vibration of carbonyl group. Identification and purity of prednisolone were interpreted three characteristic bands corresponding to -1 carbonyl group at 1622cm (α-β -1 unsaturated groups), 1688cm (aliphatic -1 group), 1707 cm (cyclic group)
The Rf value of azithromycin wwas found to be 0.867 in ethyl acetate :hexane (1:1) and Rf value of prednisolone was found to be 0.934.
Determination of absorbance maxima (λ ) of azithromycin and prednisolone max The stock solution of the drug was scanned in the wavelength range of 200- 400 nm using UV spectrophotometer. The absorption maximum (λmax) of azithromycin and prednisolone was found to be 298 nm (Figure 5) and 245 nm (Figure 6).
Calibration curve (Figure 7) of different concentration of drug in phosphate buffer saline (pH 6.5) metanol vs. absorbance was found to linear and beer's lay obeyed in the concentration range of 100- 2 800μg/ml (R =0.992) (Table 4).
Calibration curve (Figure 8) of different concentration of drug in phosphate buffer saline (pH 6.5) vs. absorbance was found to linear and beer's lay obeyed in the concentration range of 10-80μg/ml 2 (R =0.999) (Table 5).
studies the results depicts there was no change in color, no lump formation
occurred in any of the mixture at different temperature & humidity conditions, when th th observed on different days (7 , 15, 30 days) interval in comparison to initial th observation on 0 day. This confirmed that both the drugs were compatible with each other as well as with excipients. (Figure 9). R values obtained from TLC f th th studies on (7 , 15, 30 days) were approximately similar to R values of pure f th drugs and gel excipients obtained on 0 day (Figure 10), predicting the compatibility of both drugs with gel excipients.
The drugs mixture were scanned over the range of 200-400nm by using UV-visible spectrophotometer. λmax for azithromycin and prednisolone was found to be 298nm & 245nm as shown in (figure 11). Overlaid spectra of azithromycin and prednisolone (figure 12).
317
318
319
Preformulation study gives brief idea about the identification (physical appearance, solubility studies, melting point, and uv spectrophotometer, IR spectra TLC, estimation of drugs). On the basis of this study we conclude that the combinations of both drugs (azithromycin and predinsolone) can be use in the management of skin diseases like psoriasis.