INTRODUCTION:

More than 40% of active substances during formulation development by the pharmaceutical industry are poorly water soluble¹. Poor water solubility, which is associated with poor dissolution characteristics. Dissolution rate in the gastrointestinal tract is the rate limiting factor for the absorption of these drugs, and so they suffer from poor oral bioavailability². For BCS class II-drugs, the dissolution rate is the limiting factor for the drug absorption rate. An enhancement in the dissolution rate of these drugs can increase the blood-levels to a clinically suitable level.

Several techniques are commonly used to improve dissolution and bioavailability of poorly water-soluble drugs, such as size reduction ³, the use of surfactants⁴, the formulation of solid dispersions ⁵, complexation with cyclodextrins, and the transformation of crystalline drug to amorphous state⁶.

In addition to the general solubility enhancement techniques described above, drug particle size reduction has often been used, in regards to the Noyes–Whitney and Ostwald–Freundlich equations, to enhance dissolution of poorly water soluble compounds ⁷.

Many approaches have been attempted to produce microparticles, including milling⁸, supercritical fluid technique ⁹ and solvent change method ¹⁰. Physical methods such as milling and grinding are successful in particle size reduction; however the particle size uniformity is not achieved and extremely inefficient due to high energy input . Disruptions in the crystal lattice can cause physical or chemical instability.

Micronized powders with a higher energetic surface show poor flow property and broad size distribution. Supercritical fluid technique is believed to be attractive methods for the size reduction, providing particles with narrow size distribution. However, they also have the limitations of low yield and high equipment cost ¹¹. Therefore, in recent years, solvent change method (antisolvent precipitation method) has been used for microcrystallization of drugs in the presence of excipients for increasing the dissolution rate of poorly water soluble drugs . Particle size reduction is achieved because adsorption of excipients onto the particle surface that inhibits particle growth¹². Crystal morphology may be altered by preferential adsorption of stabilizing agent onto specific faces of the growing crystal¹³. Powder wettability can be increased through adsorption of hydrophilic stabilizing agent. Thus it is clear that precipitation in the presence of stabilizing agent can have a positive effect on dissolution rate. This technique is a rapid, easy to handle, needs only common equipment and direct process, which can be performed with ease.

Tinidazole is an anti protozoal, anti bacterial agent used in the treatment of amoebiasis, trichomoniasis and giardiasis. Tinidazole is a BCS-class II drug, due to which dissolution rate is the rate limiting step for its absorption. The aim of this study is to prepare and characterize tinidazole microcrystals and optimize the stirring speed and stabilizing agent concentration.

MATERIALS AND METHODS:

Materials:

Tinidazole was obtained as gift sample from Dr.Reddys Pharmaceuticals (Hyderabad, India). Povidone (PVP), polyethylene glycol (PEG 400) and hydroxy propyl methyl cellulose (HPMC) were procured from Glenmark (Mumbai, India). Methanol and hydrochloric acid were of AR grade (Qualigens, Mumbai, India).

Methods:

Preparation of Microcrystals:

Microcrystals were prepared by emulsion solvent diffusion method. A weighed amount of drug was dispersed homogenously in 10 ml of methanol. This organic phase was added at room temperature, under constant mechanical stirring (200rpm) to 100 ml of 0.5% w/v aqueous solution of surfactants (PVP, PEG-400, and HPMC). Stirring was continued for 30 minutes. Microcrystals were separated by filtration using Whatman filter paper no.1 and dried for 24 hrs at Room temperature. From this best formulation was selected and for those different stirring speeds were maintained (400rpm, 600rpm).

Table.No:1 List of formulae used to prepare microcrystals

S. No.	FORMULATION	CODE
1	Drug + PVP,200rpm (1:1)	MC-1
2	Drug + PEG-400,200rpm(1:1)	MC-2
3	Drug+HPMC,200rpm(1:1)	MC-3
4	Drug + HPMC, 400rpm(1:1)	MC-4
5	Drug + HPMC, 600rpm(1:1)	MC-5

Evaluation:

Fourier Transform Infrared Spectroscopy (FT-IR) studies:

FTIR Spectroscopy was performed on each at the samples to determine the structure of the organic compounds and to identify the presence of specific functional groups within a sample. Furthermore drug polymer interactions were examined using the resulting spectra. The infrared spectra were obtained using a scale of wave numbers (cm^-1). The analyses were performed by using a thermo Nicolet nexus 470 FTIR ESP.3-5mg of sample was added to approximately 100mg of KBr. The mixture was then ground to a fine powder using a mortar & pestle and transparent discs formed using a pellet press. The discs were placed in FTIR spectroscopy apparatus and spectra were collected.

Particle size determination:

Particle size determination was carried out using optical microscopy with a calibrated eye piece micrometer and stage micrometer by taking a small quantity of formulation on slide. About 100 microcrystal size was measured individually, average was taken and their size range and mean diameter frequency was calculated.

Average Particle size is calculated by the formula,

Average Particle size= εnd/ n

Solubility studies:

Pure drug (50mg), MC-1, MC-2, MC-3, MC-4 and MC-5 under test was placed in a test tube containing 10ml distilled water. The samples were shaken at room temperature until equilibrium was achieved and the aliquots were filtered. The filtered samples were diluted suitably and assayed spectrophotometrically at 310nm.

Drug content uniformity:

From each batch of the prepared microcrystals 50mg were taken and analyzed for drug content. 50mg of microcrystals was weighed and taken into a 50 ml volumetric flask; Methanol was added to make up the volume to 50 ml and mixed the contents thoroughly and kept aside for 4 hrs with occasional shaking to facilitate the extraction of drug from the solid mixture into solvent. The solution was filtered and diluted further with methanol and assayed for their drug content spectrophotometrically by measuring absorbencies at 310nm.

Dissolution studies:

The release of Tinidazole form Microcrystals was investigated in 0.1N Hcl as a dissolution medium (900ml) using the paddle method specified in USP X XIV (model TD T6P-Electrolab).sample of 50mg Microcrystals were taken in the dissolution flask. A speed of 50 rpm and temperature 37+ 0.5° C was maintained through out the experiment. At fixed intervals, aliquots (5ml) were withdrawn and replaced with fresh dissolution media.

The concentration of drug released at different time intervals was then determined by measuring the absorbance using visible spectrophotometer at 310nm against blank.

The studies were carried out in triplicate.

The basic Invitro release data was tabulated and graphed as

· Cumulative percent drug released Vs time.

· Log cumulative percent drug retained Vs * T

· Log cumulative percent drug released Vs log time.

RESULTS AND DISCUSSION:

Preparation of Microcrystals:

The emulsion solvent diffusion method was used as a method of choice for the preparation of the microcrystals of Tinidazole. The method was simple and efficient because it does not consume energy for homogenization. The method includes the formation of microcrystals by addition of organic phase containing drug to aqueous solution of the surfactants by using a syringe through 16 gauge needle. Methanol as selected as the organic phase. Stirring at 600 rpm using mechanical stirrer was optimized and found to be efficient to get smaller particle size microcrystals. PEG400, PVP and HPMC were selected as stabilizers. Concentration of the surfactants was kept constant.

The dispersion of drug and methanol added gradually in the aqueous phase containing dissolved polymer, and the added droplets solidified into the microcrystals. It was found that the preparations of microcrystals were controlled by two processes, drug-polymer complexation and solidification. The combined effect of stirring and stabilizers result in reduction of size and increased hydrophilic characters of the drug. The solidified crystals were dried at room temperature. The manufacturing of a microcrystals implies the creation of additional surface area and hence interface. As the Gibbs free energy change, associated with the formation of additional interface is positive, the microcrystals formed are thermodynamically unstable and will tend to minimize their total energy by agglomeration. Kinetically, the process of agglomeration depends on its activation energy. This activation energy can be influenced by adding stabilizers to the system. A first requirement for a stabilizing system is that it provides wetting of the hydrophobic surfaces of the drug particles.

Fourier Transform Infrared Spectroscopy (FT-IR) studies:

The drug and polymer interaction studies showed that there is no change in their physicochemical property during time of work. Hence, the polymers tested could be taken for further studies. In FTIR study, Tinidazole showed same characteristic bands between the same peaks were identified in the drug blended with polymers (PVP, PEG-400 and HPMC). The FTIR spectra of the physical mixture of the drug with polymers exhibited all the characteristic bands as in the spectrum of the individual Tinidazole, PVP, PEG-400 and HPMC excluding the possibility of any interaction, chemical and functional group change during the processing of the formulation of microcrystals is ensured.

Particle Size Determination:

Particle size determination was carried out using optical microscopy with a calibrated eye piece micrometer and stage micrometer by taking a small quantity of formulation on slide. About 100 microcrystal size of optimized formulation was measured individually, average was taken and their size range and average mean diameter was calculated and shown in the Table No.2

Figure No.1 FTIR spectra of pure drug Tinidazole physical mixture

Figure No.2 FTIR spectra of pure drug and HPMC

Figure No.3 FTIR spectra of pure drug and PVP physical Mixtur

Figure No.4 FTIR specta of pure drug and PEG-400 physical mixture

Table.No:2 Particle Size determination of Tinidazole Microcrystals

S. No.	Microcrystals	Average particle size
1	MC-1	332.4 µm
2	MC-2	356.8 µm
3	MC-3	278.2 µm
4	MC-4	224.6 µm
5	MC-5	201.2 µm

Drug Content Uniformity

The drug content was found to be good among the different batches of the prepared samples and ranged from 73.73% to 99.19 % (Table-3 ) The drug content of the pure drug and microcrystals(MC-1,MC-2,MC-3,MC-4,MC-5) was found to be 47.04%,98.69%,73.73%,91.65%,98.94%,99.19%. In comparision between three surfactants used in the preparation of microcrystals, HPMC shows maximum drug content than PVP and PEG.. So HPMC surfactant is used further to study speed effect. Microcrystals prepared with HPMC at 600rpm(MC-5) were proved more efficient of all used polymers due to decrease in particle size by size reduction. This results in increase in surface free energy leads to increase in drug content.

Table.No:3 Solubility Profiles of Tinidazole Microcrystals

S. No.	Microcrystals	Solubility (mg/ml)
1	Pure drug	15.520
2	MC-1	33.019
3	MC-2	24.543
4	MC-3	30.4515
5	MC-4	32.9626
6	MC-5	33.0648

Solubility studies

As water is a universal solvent, apparent solubility studies were carried out in deionised water. In solubility studies of the samples, the microcrystals prepared using HPMC at 600rpm have showed highest solubility of the drug in water (33.0648 mg/ml) as compared with the untreated drug (15.520 mg/ml). Microcrystals prepared with HPMC at 600rpm (MC-5) were proved more efficient of all used polymers due to decrease in particle size by size reduction. This results in increase in surface free energy leads to increase in solubility.

Figure No.5 Photographs of Microcrystals prepared with HPMC

Figure No. 6 Photographs of Microcrystals prepared with PVP

Figure No.7 Photographs of Microcrystals prepared with PEG-400

Figure No.8 Photographs of pure tinidazole drug

In-Vitro Dissolution Studies:

The dissolution studies were carried out in 0.1N HCL which is mentioned as USP dissolution media. The drug microcrystals prepared with polymers exhibited better dissolution rate when compared with plain drug, which indicates the deposition of polymer on the drug surface. The dissolution profile of the pure drug and the polymeric microcrystals explains that the particle size reduction was an effective and versatile option to enhance the rate of dissolution. The microcrystals prepared without surfactant shown 82.878% dissolution after completion of 45 minutes, while that with HPMC,PVP,and PEG-400 shown 99.52%, 8 and 97.89% ,91.12% dissolution after completion of 30 minutes. Whereas with HPMC at 400rpm shown 99.96% dissolution after 20 minutes and HPMC at 600rpm had shown 99.98% dissolution after completion of 15 minutes. Microcrystals prepared with HPMC at 600rpm were proved more efficient of all used polymers, Due to decrease in particle size by size reduction. This results in increase in surface free energy leads to fast release.

Figure No.9 Comparative Invitro Release Profile of Tinidazole from Microcrystals

Kinetics:

In order to elucidate the release mechanism, the data of MC-5 were fitted into the models representing zero order, first order, Higuchi and korsemeyer’s equations.

When data was plotted according to zero order kinetics, a linear plot was obtained with their high regression coefficient value 0.9969, suggesting that the rate of release from microcrystals was followed as per “zero order kinetics”.

The data fitted with higuchi equation yields a linear plot with their high regression coefficient values 0.91555,indicating that mechanism of release from microcrystals was diffusion controlled .To know precisely whether fickian’s or non fickian’s diffusion exists the data was plotted according to Korsemeyer equation . The plot showed the slope value n=1.77760, this shows that mechanism of release was “Super case-II transport”

ACKNOWLEDGEMENT:

The authors wish to thank Dr.Reddy’s LABORATORIES, Hyderabad for supplying gift samples of pure drug required for our research work. The authors are thankful to PRRM College of pharmacy, Kadapa for their valuable support in carrying out this work.

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Received on 05.08.2011 Accepted on 18.08.2011

Asian J. Pharm. Tech. 1(3): July-Sept. 2011; Page 64-69