Formulation of Azithromycin Suspension as an Oral Dosage Form

Azithromycin is the drug of choice in the treatment of several bacterial infections, most often those causing middle ear infection, bronchitis, pneumonia, typhoid and sinusitis. It’s also effective against certain urinary tract infections and venereal diseases. This study was carried out to prepare an acceptable suspension either as dry physical mixture powder or granules to be reconstituted, through studying the effect of various type and concentration of suspending agent (xanthan gum, hydroxypropyl methylcellulose (HPMC), either alone or in combination) on the release profile of the drug. The best prepared suspension formulas (H& III) were selected depending on the dissolution profile of each formulas and then compared with the reference suspensions (Zithromax and Azi-once).The viscosity, sedimentation volume, Resuspendability and expiration date were evaluated for the chosen formulas (H&III) and compared with references Zithromax  and Azi-once  .The result indicated that the chosen formula – H had better dissolution rate compared with references suspensions, Also it was less viscous than them.While other chosen formula – III had lower dissolution rate compared with Zithromax  and higher dissolution rate than AZi – once, also it was less viscous than both references.It was found that the dry physically mixed powder (formula – H) was more stable than the granular suspension (Formula III) since the expiration date for formula H and formula III were 3.24 and 2.7 years respectively.


Introduction
Suspension is preparation containing finely divided drug particle (Suspensoid) distributed uniformly throughout a vehicle in which the drug exhibits a minimum degree of solubility (1) .Some suspensions are available in ready liquid form that is, already distributed through a liquid vehicle with or without stabilizers and other additives.This preparation is termed as an "Oral Suspension "in the united state pharmacopeia (USP).Other preparations are available as dry powders to be reconstituted when desired.This preparation is termed as "For Oral Suspension "in USP (1)   .There are many physical and chemical consideration in the preparation and development of a suspension to satisfy its pharmaceutical requirements.Some suspending agents are generally added to the dispersion medium in order that their structures help to maintain uniform dispersibility (2) or to prevent caking of the drug particles during shelflife (3) .Pharmaceutical suspension usually defined as a coarse dispersion (4) .
Azithromycin is one of the worldś best selling antibiotics, and is derived from erythromycin.Azithromycin is used to treat or prevent certain bacterial infections , most often those causing middle ear infection ,tonsillitis, throat infections, laryngitis, bronchitis, pneumonia, Typhoid and sinusitis (5,6) .In recent year it has primarily been used to prevent bacterial infections in infants and those with weaker immune system.It is also effective against certain urinary tract infection and venereal diseases, such as nongonococcal urethritis, Chlamydia, gonorrhea and cervicitis (7,8)   .Unlike erythromycin, azithromycin is acid stable and can therefore be taken orally with no need of protection from gastric acids.It is readily absorbed, but its absorption is greater on an empty stomach (9) .Azithromycin is practically insoluble in water, freely soluble in anhydrous ethanol and methylene chlorid (10) .

Formulation of azithromycin suspension
Several formulas of Azithromycin aqueous suspension were prepared, either as dry physical mixture powder for reconstitution or as granules for reconstitution.

Suspension prepared by physical mixing (as dry powder)
Table (1) shows different formulas of azithromycin suspension each was prepared as: Ablend of 4.0 %( w/v) azithromycin was mixed with different excipients stated in the above table, in a dry amber bottle (11) .

Suspension prepared as granular for reconstitution
Table (2) shows different formulas of azithromycin granules prepared by granulation method to be reconstituted as suspension as follows:-A blend of 4.0 %( w/v) Azithromycin was mixed with different excipients mentioned in the above table, followed by levigating with alcoholic PVP solution (0.5% w/v) as a granulating agent.The powder mixture passed through a sieve (300µm) to a petri dish for drying at 37°C before being transferred to an amber bottles (12,13) .The evaluation of prepared suspension and comparative studies of different variables affecting the selected formulas with marketed references (Azi-once ® and zithromax ® suspension) were done.

Dissolution profile
The dissolution rate of azithromycin drug from suspensions was studied using USP dissolution apparatus rotated at 50 r.p.m.The dissolution medium was 0.1N HCl (500 ml) maintained at 37°C, 5 ml sample of suspension was added to the medium.Then a sample of dissolution medium was withdrawn at different time intervals (1, 2, 3, 4, 5, 7, 9, 12, 20, 30 minutes) through a pipette fitted with a filter paper.The same fresh dissolution medium was added to the jar each time to replace with drawn samples.Each sample was suitably diluted and assayed spectrophotometrically at 210nm (10) for azithromycin content (14) .

Measurement of viscosity
The viscosities were obtained at 37C with NDJ -55 digital rotational viscometer using spindle number 2 at 60 rpm. (15).

Sedimentation volume measurement
Fifty ml of each suspension was diluted with distilled water to a volume of 100 ml in a stopper graduated cylinder.The suspensions were shaken vigorously to ensure uniformity, and then left undisturbed.The sedimentation volume was measured every 4 hours for a period of 48 hours (16) .

Re-Suspendability of suspension
The test consisted of manually shaking the cylinder after the sedimentation experiment was completed.Based on the effort required to convert the sediment system to a homogenous suspension, the prepared product was rated as: resuspendable , resuspendable with difficulty or not resuspendable (17) .

Antimicrobial activity of suspension
The antimicrobial activity of azithromycin suspensions were studied by the agar diffusion method (18,19) .The basic of the diffusion test includes the diffusion of the material under test from a central reservoir into the surrounding agar which has been inculated with a sensitive organism.The growth of the organism is inhibited, and manifested as zone of inhibition.The relationship between the applied dose , kind of suspension and size of the inhibition zone in the basis of comparisons the gram +ve and the gram -ve bacteria which are sensitive to azithromycin were used .

The test procedure
Steril Miller -Hinton agar media were poured in to flat bottom steril petri dish under aseptic technique and were allowed to harden , then incubated at 37°C for 24 hours after inoculated with the appropriate microorganism , all of these plates are divided into four sections as shown in figure (a) (14) .0.05ml (one drop) of each suspension formulas were inoculated in each sector of the agar media using a micropipette, each drop contains the equivalent of 2mg of azithromycin.

Stability study
The accelerated stability study was done in order to determine the expiration date of formula H and formula III by placing the samples of both formulas in ovens at 40°C, 50°C and 60°C for 90 days.Samples (dry physical mixture powder or granules) were taken and assayed for their drug content at a suitable time intervals using UV spectrophotometrical method for the two accepted formulas (H and III) (20) .Furthermore, physical stability (color, odor and pH) change for the samples were also examined .

Results and Discussion
Xanthan gum was used as suspending agent because of its excellent suspending properties and also as an effective flocculating agent at relatively low concentration (21) .An increase in the concentration of xanthan gum (Formula B) gave no substantial change in flocculation behavior, good dissolution properties and it produced a sediment layer that was easily redispersed upon shaking as shown in figure (1).On the other hand, hydroxy propyl methyl cellulose (HPMC) was utilized as suspending agent and binder as shown in figure (2).This polymer behave as a protective colloid by coating the solid hydrophobic particles with multimolecular layer, this will impart hydrophilic character to the solid and thus promote wetting (22) .But to a certain concentration, the dissolution of azithromycin decreased which is attributed to the fact that at high polymer concentration, the polymeric flocculating agent coat the whole surface of the suspended particles, so there is no free areas from adsorbate.These areas are necessary for cross-linking to be recure after the product was sheared (23) .The increase in the viscosity of the 4 2 3 1 system may also have an effect on the dissolution rate of the drug (24) .Furthermore, a combination of xanthan gum and HPMC (Formula E , F, G and H ) resulted in a maximum enhancement in the dissolution of azithromycin , this could be due to the fact that their linear branched chain molecules form a gel like net work within the system and become adsorbed on to the surface of the dispersed particles , thus holding them in a flocculated state ( bridging effect ) (20 ) as shown in figure (3)  .Formula -H produced a suspension with good dissolution as shown in figure (3).This formula (H) gave the most optimum physical stability and remarkable release profile, therefore it was chosen for extensive study and to be compared with reference suspensions.The addition of colloidal silicon dioxide as a dispersing agent (formula I&J) results in too viscous suspension as listed in table (3), with difficulty to redisperse upon shaking.This was a result of using a combination of three polymers (xanthan gum +HPMC and colloidal silicon dioxide) which leads to rheological synergism to be occurred due to stronger cross linking between these polymers (25) .These formulas (I &J) showed a substantial change in the dissolution behavior corresponding to the best formula -H and the releasing profile decreased by increasing the concentration of colloidal silicon dioxide as shown in figure (4) (26,27) .-Formula H(0.5 % w/vxanthan gum + 0.3% w/v HPMC) -Formula G (0.3% w/v xanthan gum + 0.3% w/v HPMC) -Formula E (0.3% w/v xanthan gum + 0.5% w/v HPMC) -Formula F (0.5% w/v xanthan gum + 0.5% w/v HPMC) Table 3: The viscosities of the suspension formulas ( H , III, I , J) and reference suspension, using spindle number 2 at 60 rpm.

Formulas
Viscosity (mpa.s)Azionce ® 268 Zithromax ® 240 Formula -H 233 Formula -III 199.2 Formula -I 450 Formula -J 400 Finally, azithromycin suspension (as dry physical mixture powder) when prepared using 0.5%(w/v) xanthan gum as a single suspending agent together with 0.3%(w/v) HPMC as suspending agent and binder (formula -H) resulted in a suspension that showed high sedimentation volume (0.99) and was easily redispersed upon simple shaking.Figure (5)  shows the enhancement in the dissolution of azithromycin by the addition of a blend of different concentrations of xanthangum and HPMC (polymeric flocculation agent) to the formulas I, II, III and IV.These formulas were prepared as granules using alcoholic -PVP solution as a granulating agent (28) .The granules were found to be free flowing, not bulky, and of uniform size.The size of each granule was 300 µm according to the sieve number was used (no.50) (1,17).Formula III was chosen since it gave good dissolution, stability although it produced sediment layer with simple shaking easily redispersable.Figure (6) show the dissolution profile of azithromycin suspension for formulas H & III compared with the reference Zithromax ® and Azionce ® suspensions.-Formula I (0.3% w/v xanthan gum + 0.5% w/v HPMC) -Formula II (0.5% w/v xanthan gum + 0.3% w/v HPMC) -Formula III (0.3% w/v xanthan gum + 0.3% w/v HPMC) -Formula IV (0.5% w/v xanthan gum + 0.5% w/v HPMC) The results showed that azithromycin release from formula -H was higher than that form others ( Azi-once and formula -III ) but show approximately the same release as zithromax ® .Formula -III after reconstitution showed lower dissolution profile than formula Hand zithromax ® , this may be due to the granulation process, since PVP was used as a granulating agent which is water soluble binder and has good swelling and hydration capacity.These properties result in the high viscous region surrounding the drug particle (29,30) .On the other hand the viscosities of the products are represented in table (3).The results showed that the viscosity of azithromycin suspension was shear rate dependent and increased viscosity in the following order: Azionce ® > Zithromax ® > Formula -H > Formula -III Table (4,5) show the sedimentation volume and resuspendability after settling of the chosen azithromycin formulas (H,III) and the reference suspensions after 48 hours undisturbing .The evaluation of azithromycin released as antibacterial agent was listed in table (6).

Products
F=H u / H 0 Zithromax ® 0.99 Formula -H 0.99 Formula -III 0.9 Azionce 0.6 Where H u is the ultimat height of the Sediment as suspension settle ,H 0 is the intial height the total suspension .Formula -H has less antimicrobial effect against gram positive microorganism compared to the references, while formula -III has the same activity as references.Formulas (III and H) has the same antimicrobial activity against gram negative microorganism as Azionce ® but less than zithromax ® (31) .The accelerated stability study was applied on the chosen formula at higher temperatures(40°C,50°C and 60°C) to predict the expiration date of formulas H and III .
The degradation of azithromycin in these formulas followed first order kinetics since straight lines,were obtained by plotting the logarithm of percent remaining of azithromycin versus time as shown in figures ( 7, 8) (32,33) .The degradation rate constants (k) at different temperatures were calculated from the slope of the straight lines and they were listed in table (7).Arrhenius plots were then constructed and are shown in figures (9,10) for formula H and III respectively.The Linearity of the curve indicates their utility in predicting the rate of degradation at lower temperature.The rate constant at 25  C, obtained from those plot for dry physically mixed powder (Formula -H ) and granular suspension ( Formula III) were equal to (0.09 x 10 -3 ) and (0.11 x 10 -3 )(day -1 ) respectively.Since the degradation of the drug followed first order kinetics, the expiration date t 10% at 25  C could be calculated using the following equation.
The expiration date for formula -H and formula -III were 3.24 and 2.7 years respectively.

Figure 1 :
Figure 1: Effect of xanthan gum concentration on dissolution profile of azithromycin (Formula A and B ) in 0.1 N HCl (pH=1.2) at 37 °C.

Figure 2 :
Figure 2: Effect of HPMC concentration on the dissolution profile of azithromycin (Formula Cand D) in 0.1 N HCl (pH=1.2) at 37 °C.

Figure 3 :
Figure 3: Effect of xanthan gum and HPMC on the dissolution profile of azithromycin ( Formula E,F,G and H) in 0.1 N HCl (pH=1.2) at 37 °C.

Figure 4 :
Figure 4: Effect of colloidal silicon dioxide concentration on the dissolution profile of azithromycin from the best formula -H ( Formula I and J ) in 0.1 N HCl (pH=1.2) at 37 °C.Finally, azithromycin suspension (as dry physical mixture powder) when prepared using 0.5%(w/v) xanthan gum as a single suspending agent together with 0.3%(w/v) HPMC as suspending agent and binder (formula -H) resulted in a suspension that showed high sedimentation volume (0.99) and was easily redispersed upon simple shaking.Figure(5)  shows the enhancement in the dissolution of azithromycin by the addition of a blend of different concentrations of xanthangum and HPMC (polymeric flocculation agent) to the formulas I, II, III and IV.These formulas were prepared as granules using alcoholic -PVP solution as a granulating agent(28) .The granules were found to be free flowing, not bulky, and of uniform size.The size of each granule was 300 µm according to the sieve number was used (no.50)(1,17) .Formula III was chosen since it gave good dissolution, stability although it produced sediment layer with simple shaking easily redispersable.Figure(6) show the dissolution profile of azithromycin suspension for formulas H & III compared with the reference Zithromax ® and Azionce ® suspensions.

Figure 7 :
Figure 7: Degradation curve of dry physical mixture powder of azithromycin suspension ( Formula -H ) at different temperature.

Figure 9 :
Figure 9: Arrhenius plot for expiration date estimation of azithromycin suspension (Formula -H ) at 25  C .