Pharmaceuticals can be improved by the mechanical micronization of crystals. This, however, leads to a decrease in some other properties such as flowability and packability. Therefore, it becomes necessary to process microcrystals through a series of additional steps, e.g., mixing with fillers and granulation, for ease of handling. It would be more efficient to transform the microcrystalline drugs or excipients into an agglomerated form during the crystallization process⁵.

Azithromycin, a macrolide dicationic antibiotic^6-7, which is a semi-synthetic acid-stable erythromycin with an expanded spectrum of activity and improved pharmacokinetic characteristics⁸, markedly inhibits fluid-phase endocytosis, probably by interacting with lipid membranes⁹. It was approved by the Food and Drug Administration for clinical use in 1992. It is a member of a new generation of macrolide antibiotics and has several advantages over erythromycin. The flowability and compressibility of the macrolide antibiotics are poor because of irregular crystal habit and crystal form. The oral bioavailability of Macrolide antibiotics is low which may be due to poor aqueous solubility and dissolution behavior.

The therapeutic dose of Azithromycin is relatively high. Tablet formulation of Macrolide antibiotics with higher amount of drug substance do not lend themselves to direct compression due to poor compressibility as their crystal morphology show significant impact on compression characteristics. Direct compression method in the production of high dose formulation is limited, since large quantities of directly compressible excipients are ordinarily

required to produce suitable tablets which increase the weight and cost of the formulation.

The present study was aimed to prepare the recrystallized agglomerates of azithromycin by using neutralization technique with different inert polymers like Hydroxy Propyl Methyl Cellulose (HPMC), Polyethylene Glycol (PEG-6000), and Surfactants like Poloxomer.The generated recrystallized agglomerates were evaluated for different physicochemical properties and characterized for polymorphic or habit modification using a multitude of analytical techniques like differential scanning calorimetry (DSC), powder X-ray diffraction (XRPD), Fourier transforms infrared spectroscopy (FTIR) and scanning electron microscopy(SEM).

MATERIALS AND METHOD:

Materials:

Azithromycin was procured as a gift sample from Alembic Research Center (Vadodara, India). Hydroxy Propyl Methyl Cellulose (HPMC) was supplied by Colorcon, (Goa, India). Polyethylene Glycol (PEG-6000) and Surfactant like

Poloxomer, Chloroform, hydrochloric acid and Sodium Hydroxide were purchased from S. D. Fine (Mumbai, India).

Method:

In 50ml of 0.1N HCl, 5 gm of Azithromycin had been dissolved in a cylindrical vessel. The aqueous solution (100ml) of hydrophilic polymers or surfactants with 50 ml 0.1N NaOH were added during stirring at 500 rpm to neutralize the HCl solution of Azithromycin and crystallize out the azithromycin drug with the polymers or surfactants. Then 5 mL chloroform was added drop wise at the rate of 1 mL/min followed by agglomeration of azithromycin crystals within 45 minutes. Then, the prepared agglomerates were filtered to separate the recrystallized agglomerates from the preparation system. The resultant product was washed with distilled water and dried in an oven at 40⁰C for 12 hr. The whole process was carried out at room temperature.

Yield and drug content:

The prepared agglomerates were weighed after drying, and the production yield was calculated. Prepared agglomerates were powdered, from which powder equivalent to 20mg of Azithromycin were taken separately and extracted with 80ml of 0.1N HCl by sonicating for 20 minutes. Place few minute in refrigerator filter and adjust the pH in the range of 6-7 and make up the volume upto 100ml with distilled water. Take 1ml form the above solution diluted to 6ml with the distilled water in 10 ml volumetric flask to it add 0.5ml of Eosin Y solution (4 X 10-3 M), mix well then to it add 1 ml 0.4M acetate buffer (pH 3) adjust the volume up to 10ml with distilled water. The absorbance was measured spectrophotometrically (Pharma spec 1700, Shimadzu Corporation, Kyoto, Japan) at 544.5nm, against an appropriate blank prepared simultaneously.

Saturation solubility studies:

An excess amount of azithromycin and their recrystallized agglomerates were added in screw capped test tubes with fixed volume (20 ml) of distilled water.

The resulting suspension was treated at room temperature with 100 rpm in incubator shaker. Samples were drawn after 48 hrs and filtered through 0.2µ filters. Take 5 ml of filtrate from dispersion of above macrolide antibiotics in water after 48 hr and diluted to 60ml with 0.1N HCl, adjust the pH 6 to 7 by adding 0.4M NaOH solution and make up the volume up to 100ml with distilled water. Take 1ml form the above solution diluted to 6 ml with the distilled water in 10 ml volumetric flask to it add 0.5 mL Eosin Y solution (4 X 10-3 M), mix then well to it add 1 ml 0.4M acetate buffer (pH 3) adjust the volume up to 10ml with distilled water. The absorbance was measured spectrophotometrically at 544.5nm against an appropriate blank prepared simultaneously. Solubility of the recrystallized agglomerates in distilled water was determined either from the calibration graph or using the corresponding regression equation.

Table: 1 coding for recrystallized agglomerates of Azithromycin with hydrophilic Polymers and surfactants.

Sr.No:	Polymer or surfactants used	Agglomerated crystal code
1	Azithromycin (API)	ATM
2	Without Polymer or surfactants	ATM-Agg
3	Hydroxypropyl methyl cellulose (HPMC)	ATM-HPMC
4	Polyethylene Glycol (PEG)	ATM-PEG
5	Poloxomer	ATM-POL

Table: 2 Evaluation parameters of RTM and prepared recrystallized agglomerates with different polymers.

Sr.No.	Name of parameters*	Product Code
Sr.No.	Name of parameters*	ATM	ATM-Agg	ATM-HPMC	ATM-PEG	ATM-POL
1	Product yield (%)	NA	88 ±1.256	85 ±2.456	82 ± 2.489	84 ±1.896
2	Drug content (%)	98 ±2.586	93 ±3.458	92 ±3.158	90 ±2.678	88 ±1.859
3	Saturation solubility(mg/mL)	1.34 ±0.197	2.94 ±0.188	3.47 ±0.127	3.18 ±0.184	4.91 ±0.145
4	Bulk density(gm/mL)	0.518 ±0.026	0.263 ±0.023	0.275 ±0.042	0.260 ±0.038	0.285 ±0.068
5	Tap density(gm/mL)	0.688 ±0.073	0.312 ±0.028	0.326 ±0.018	0.310 ±0.027	0.330 ±0.028
6	Compressibility index	24.7 ±0.456	15.7 ±0.357	15.6 ±0.159	16.1 ±0.267	14.9 ±0.149
7	Hausnar ratio	1.328 ±0.024	1.186 ±0.026	1.185 ±0.028	1.192 ±0.019	1.175 ±0.025
8	Angle of repose	40 ±0.986	25 ±0.896	18 ±0.987	23 ±0.879	23 ±0.689
9	Wettability study (Water raising time-hrs)	19 ±0.156	11 ±0.265	09 ±0.245	11 ±0.345	07 ±0.125
10	Packability parameters from Kawakita and Kuon equation
	a	0.364	0.218	0.191	0.213	0.231
	b	0.0055	0.0119	0.0087	0.0100	0.0121
	k	0.0104	0.0071	0.0062	0.0054	0.0029

* Each value represents mean ± S.D. (n = 3)

Table: 3 principal or characteristic FTIR peak of ATM and their recrystallized agglomerates.

Sr.No	Crystal code	Principal peak (cm^-1)
1	ATM	1726.35	1182.40	1053.17
2	ATM-Agg	1726.35	1182.40	1053.17
3	ATM-HPMC	1726.35	1182.40	1053.17
4	ATM-PEG	1726.35	1182.40	1053.17
5	ATM-POL	1720.56	1186.26	1051.24

Figure: 1 FTIR spectra of Azithromycin and their recrystallized agglomerates by neutralization technique.

Flow properties:

Flow properties of the drug and prepared recrystallized agglomerates were studied by determining the bulk density (σ_b), tap density (σ_t), Carr’s Index and Hausner ratio. A weighed quantity of the samples was taken to determine the bulk and tap density. The parameters were determined using following equations.

Bulk density (σ_b) = Mass / Poured volume (1)

Tap density (σ_t) = Mass / Tapped volume (2)

Carr’s Index = [(σ_t – σ_b) / σ_t] x 100 (3)

Hausner ratio = (σ_t / (σ_b) (4)

Angle of repose (Fixed funnel and free standing cone method):

A funnel with the end of the stem cut perpendicular to the axis of symmetry was secured with its tip 2.5 cm height, H, above graph paper placed on a flat horizontal surface. The powder samples were carefully poured through the funnel until the apex of the conical pile so formed just reached the tip of the funnel (H). The mean diameter (2R) of the powder cone was determined and the tangent of the angle of repose was given by:

Values for angle of repose ≤ 30⁰ usually indicate free flowing material and angle ≥40^O suggested a poor flowing material. The angle of repose can be obtained from the equation:

Tanθ = h/r, q = tan ^–1 (h/r)

Tanθ = h/0.5d,

Where

q = Angle of repose

h = height of the tip of funnel from horizontal plane

r = radius of the pile made by powder

D-diameter of cone.

Measurement of Packability:

The packability of the samples was investigated by tapping them in a 25-ml measuring cylinder using a tapping machine. Initially, 10 gm of substance was weighed and then gently poured into a measuring cylinder. The volume of 10 gm sample was recorded. The poured density (minimum density) was calculated from the powder mass (25 g) and the volume. Then the cylinder was tapped and the volume was recorded after every 100 taps until the volume did not change significantly. The compressibility was evaluated by measuring the tapped density according to the modified Kawakita (I) and Kuno (II) equation

N/C = 1/ (ab) + N/a......I

Where as {C = (Vo-Vn)/Vo, a = (Vo-V∞) /Vo.}

N =Number of tapping, C =Difference in volume (degree of volume reduction.), a and b = constant for packability and flowability, Vo = Initial volume, Vn = Final volume after n^th tapping, V∞ = Powder bed volume at equilibrium.

ρ_f- ρ_n = (ρ_f- ρ_o) . exp. (-kn)…II

Where as ρ_f, ρ_o, ρ_nApparent densities at equilibrium, nth tapped, initial state respectively

The compressibility was assessed by comparing the constants a, b and k in Eqs. I and II, respectively. The constant a represents the proportion of consolidation at the closest packing attained and constant 1/b describes cohesive properties of powders or the apparent packing velocity obtained by tapping. The constant k in Kuno’s equation represents the rate of packing process.

Wettability/ powder bed hydrophilicity study:

The untreated azithromycin and prepared recrystallized agglomerates were placed on a sintered glass disk forming the bottom of glass tube on which methylene blue crystals were placed. The whole device was brought into contact with water. Measure the time taken for the capillary rising of water to the surface so as to dissolve methylene blue crystals was noted. Minimum is the time required to reach the water to surface maximum is its wettability.

In-vitro dissolution studies:

The in vitro dissolution studies were carried out using eight station USP type II dissolution apparatus. The study was carried out in 900 ml Acetate buffer pH 5.0 as dissolution medium. Dissolution medium was kept in a thermostatically controlled water bath, maintained at 37±0.5⁰C temperature. The paddle was rotated at 100 rpm. At predetermined time intervals, 5ml of samples were withdrawn and assessed for drug release spectrophotometrically.

Figure: 2 FTIR spectra of Azithromycin and their optimized recrystallized agglomerates by neutralization technique.

Take 1ml quantity of withdraw sample into 10ml volumetric flask and diluted to about 6ml with distilled water. to it add 0.5mL Eosin Y solution (4 X 10-3 M), mix then well to it add 1 ml 0.4M acetate buffer (pH 3) adjust the volume up to 10ml with distilled water. The absorbance was measured spectrophotometrically at 544.5nm against an appropriate blank prepared simultaneously. At each withdrawal, 5ml of fresh dissolution medium was added to dissolution jar so as to maintain sink condition. These samples were assayed through ultraviolet absorbance measurement at 544.5nm using UV-Visible Spectrophotometer (Shimadzu UV-1700, Japan) by an analytically validated method (r² = 0.9992). To increase the reliability of the observations, the dissolution studies were performed in triplicate.

Figure: 3 DSC of Azithromycin and their optimized recrystallized agglomerates by neutralization technique.

Figure: 4 Dissolution profile study of Azithromycin and their recrystallized agglomerates.

Figure: 5 SEM studies of Azithromycin and their recrystallized agglomerates.

Differential scanning calorimetric studies

Differential scanning calorimetric (DSC) analyses of the samples were carried out by using differential scanning calorimeter equipped with computer analyzer (Shimadzu TA –60 differential scanning calorimeter, Shimadzu Corporation, Kyoto, Japan). Samples (of 3-7 mg) were heated under nitrogen atmosphere on an aluminum pan at a heating rate of 10 °C/min over the temperature range of 20-300^oC.

Powder X-ray diffraction studies:

Powder X-ray diffraction (PXRD) patterns were traced employing X-ray diffractometer (Philips PW 1729, Analytical XRD, Holland) for the samples using Ni filtered CuK(α) radiation (intensity ratio(α₁/ α₂): 0.500), a voltage of 40 KV, a current of 30 mA and receiving slit of 0.2 inches. The samples were analyzed over 2q range of 5.010-39.990^o with scanning step size of 0.020^o (2q) and scan step time of one second. To minimize the effect particle size on preferred orientation, all the samples were passed through sieve #120 and collected on sieve # 240 (# 120/240).

Fourier transforms infra red spectroscopy (FTIR):

FT-IR spectra of prepared spherical agglomerates along with the drug and drug with excipients were recorded on Shimadzu FT IR – 8400 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Potassium bromide pellet method was employed and background spectrum was collected under identical situation. Each spectrum was derived from single average scans collected in the region 600 – 4000 cm^-1 at spectral resolution of 2 cm^-2 and ratioed against background interferogram. Spectra were analyzed by software supplied by Shimadzu.

RESULTS AND DISCUSSION:

DSC Study:

The azithromycin DSC curve (figure: 3) shows sharp endothermic peak at 108.250C indicating its melting point. The optimized recrystallized agglomerates with HPMC (ATM-HPMC) and poloxomer (ATM-POL) shows endothermic peak at 82.120C and 95.190C respectively. This indicated that decrease in melting point due to conversion of polymorphic form and crystal habit.

XRD Study:

The figure: 2 represent the XRD Spectra of azithromycin and recrystallized agglomerates. The maximum disappearances of peaks were observed in the recrystallized agglomerated crystals. There is difference in d-spacing between the XRD spectra of azithromycion and the recrystallized agglomerates at respective 2θ angle referring the habit modification and change in the intensity of peaks, which indicate different arrangement of the molecules hence confirming the development of different polymorphic form.

FTIR Study:

In the range of 4000 to 500 cm^-1azithromycin exhibit a strong sharp signal at 1726 cm^-1which is due to absorption of the carbonyl group. From the literature survey the principal or characteristic peak of Azithromycin FTIR includes 1721, 1188, 1052cm^-1. The spectrum of recrystallized agglomerates (figure: 1 and table: 3) corresponds to the superimposition of raw crystals of azithromycin (mentioned in table and figure) with no significant shift of major peaks. The close agreement between the FT-IR spectra of the raw azithromycin crystals and their recrystallized agglomerates suggested that there were no changes in the ATM molecular structure caused by the recrystallization and the agglomeration process. This confirms the presence of azithromycin in recrystallized agglomerates without changing the chemical structure.

SEM study:

SEM Study was conducted to observe the crystal habit of Azithromycin and their recrystallized agglomerates. The SEM study (figure: 5) revealed that the ATM is irregular stone shaped crystals where as the prepared recrystallized agglomerates are aggregates of recrystallized crystals which show irregular spherical shape.

Production yield and drug content:

The production yield of the prepared recrystallized agglomerates by neutralization technique shows above 80 % which was calculated by measuring the weight of the prepared recrystallized agglomerates by considering the raw material taken for the spherical crystallization as 100.0%. The production yield of recrystallized agglomerates by neutralization were on lower side in between 80-90%.The content in the prepared recrystallized agglomerates shows in range of 88-96% mentioned in table:2 which is lower then that of raw crystals of azithromycin might be due to loss of 4-5% drug during processing. The reason for loss of drug may be due to sticking of drug to the walls of the beaker or to the stirrer shaft.

Saturation solubility:

The solubility study was carried out in distilled water for both Azithromycin and their recrystallized agglomerates with different hydrophilic polymers. Solubility in distilled water for ATM (1.34 ±0.197 mg/mL), and recrystallized agglomerates like ATM-Agg (2.94 ±0.188) ATM-HPMC (3.47 ±0.127 mg/ml), ATM-PEG (3.18 ±0.184 mg/ml), and ATM-POL (4.91 ±0.145 mg/ml). Significantly improved (** P<0.01) the solubility of recrystallized agglomerates in distilled as per mentioned in table: 2. This may be due to by changing the crystal forms because different habit, structure, surface modification and in some instances, solvents included into the crystals forms solvets or clathrates can changes the surface properties and the reactivity of drug particles and internal energy of the molecules play an important role to increase solubility.

Powder bed hydrophilicity study:

Table: 2 indicate powder bed hydrophilicity study of ATM and their recrystallized agglomerates. The recrystallized agglomerates showed significantly shortest rising time (_** P<0.01) of water to its surface as compared to raw ATM crystals represent better wattability of prepared granules as compared to raw FNO. The order of wettability was ATM-POL> ATM-HPMC>ATM-Agg, ATM-PEG>ATM. The reason for the superior wettability with poloxomer is due to adsorption of it on the raw crystals of ATM during preparation.

Flowability parameter:

Table:2 represents the bulk, and tap density of the agglomerated crystals, the results indicated that both the densities of the agglomerated crystals showed decrease value because of the increased in volume and the total porosity of agglomerated crystals. The ATM has higher bulk density and thus lower porosity as compared to the prepared recrystallized agglomerates. The lower density is likely to be related to the intraparticle porosity or particle density and hence the reduction in bulk density of the recrystallized agglomerated samples indicates a greater porosity within the agglomerated particles.

Table: 2 Represents flowability parameters of the ATM and recrystallized agglomerated crystals in term of Angle of repose, Carr index and Hausnar ratio. Recrystallized agglomerated crystals were found to have significantly lower angle of repose (** P<0.01) in comparison to the raw crystals of azithromycin , which could be due to the irregular stone shaped crystals of azithromycin that is reflected from SEM Fig.5 which hindered in the uniform flow of crystals from funnel. The reason for the excellent flowability of recrystallized agglomerated crystals was due to significant reduction in interparticle fraction because of their agglomerated spherical shape with reduction in the surface area. The Carr index revealed that the flowability of the azithromycin was significantly poor (** P<0.01) then that of the agglomerated crystals i.e. these agglomerates were lower Carr index then raw crystals. Hausnar ratio of agglomerated crystals was less then a raw crystal indicates improvement in flowability of agglomerated crystals.

Packability study:

Packing process of the recrystallized agglomerates in a measuring cylinder by tapping was described by Kawakitas and Kunos equation. Agglomerates were easily packed by tapping, the process of which was evaluated based on percent compressibility and parameters of the Kawakita equation. The packing ability was assessed by comparing the constants a, b and k in Kawakitas and Kunos equation (figure: 2). The constant a for the agglomerated crystals was smaller then the raw unagglomertaed crystals of ATM. This indicated that the agglomerated crystals were easily packed, even without tapping. The larger b values of the agglomerated crystals proved that the packing velocity of the agglomerated crystals by tapping was slower then that of the crystals which are not agglomerated. The smaller k in kuno equation for the agglomerated crystals coincidence with the above findings. The increasing packability of agglomerated crystals may be due to lower surface and wider particle size distribution of spherical crystals, during tapping process smaller particle might have infiltrated into the voids between the larger particles and resulted in improved packability (10, 11).

Dissolution study:

Figure: 4 represent the dissolution of ATM and their recrystallized agglomerated. It was observed that the ATM shows 58 % cumulative drug release with in 45 minutes. The prepared recrystallized agglomerated crystals with hydrophilic polymers and surfactant were showed significantly (**P<0.01) improvement in % CDR at 45 minutes comparative to the raw crystals of ATM. The dissolution profile of ATM exhibit better dissolution behavior for recrystallized agglomerates then raw crystals. The reason for this faster dissolution could be linked to the adsorption of polymers during recrystallization on the crystal surface and change in crystal habit and crystalline form. The reason for the improvement in dissolution was also due to better wettability of the recrystallized agglomerates.

CONCLUSION:

Neutralization technique can be applied to produce recrystallized agglomerates of azithromycin with modified properties. The chances of entrapment of organic solvent (Residual solvent) is mostly eliminated because the whole process was carried out with minimum use of organic solvent. Azithromycin recrystallized agglomerates with hydrophilic polymers and surfactant produced in this investigation showed dramatically improved physicochemical properties such as solubility, dissolution, flowability, packability, and wettability. The process is the simple that is also inexpensive enough for scaling up to a commercial level; this reduces time and cost by involving faster operation, less machinery and fewer personnel with great advances in tabletting technology.

ACKNOWLEDGEMENTS:

The authors express their gratitude to Alembic Research Center (Vadodara, India) for providing Azithromycin as gift sample for this research work.

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