INTRODUCTION:

Modern pharmaceutical research and development acknowledge the great importance of the relationship between crystal structure and physical properties of drugs. Different crystalline forms of drugs, as well as amorphous forms, have different physical properties that may affect both stability and bioavailability. In this respect, the investigation for polymorphic phases is a mandatory requirement during very early stages of solid-state characterization on new drugs¹.

Pharmaceuticals can exist in various solid forms having different physical and chemical properties. These solid forms include ‘true polymorphs’, solvates, desolvates and amorphous solids. This concept of variability broadly classified as polymorphism hereafter, was first introduced by Klaproth in 1788, who identified the two different forms of calcium carbonate as calcite and aragonite². Polymorphism has contributed significant variability in product performance in pharmaceutical, chemical and food industry and continues to pose a challenge to pharmaceutical scientists in producing drugs of consistent quality. It also provides a unique opportunity, to engineer solids having ‘tailor made’ properties (challenges).

Surprisingly, a very large number of pharmaceuticals exhibit the phenomenon of polymorphism. 70% of barbiturates, 60% of sulfonamides and 23% of steroids exist in different polymorphic forms³. Those who study polymorphism are rapidly reaching the conclusion that all compounds, organic or inorganic, can crystallize in different solid forms.

Crystallization is a separation and purification technique commonly used in the chemical industry which involves diffusional process accompanied by formation of new heterogeneous phase. Many chemical substances can crystallize in different crystal forms a phenomenon known as polymorphism. Polymorphs are defined as crystalline phases that have different arrangements and/or conformations of molecules in the crystal lattice. From a pharmaceutical point of view, bioavailability, the ability to process and stability of the product are influenced by the existence of varied physical and chemical properties of these solid-state forms³. Supersaturation, nucleation and crystal growth are the predominant physical phenomena associated with crystallization ⁴.

Supersaturation is defined as the concentration of the solute in excess of saturated concentration under given conditions of temperature. It is composed of two zones. The metastable zone shows crystal growing without nucleating, whereas unstable region crystals appear after nucleation ⁴.

Nucleation is first step in crystal formation where molecules collide with each other in solution leading to pre-nucleating clusters. As population of clusters increases they begin to associate to form an embryo. Some embryos through additional collision grow into nuclei contributing to formation of macroscopic crystal which is known as crystal growth ⁵.

Fig.1. Scanning electron micrographs of:

(1) untreated nimesulide, nimesulide crystallized from methanol as: (2) At cool temperature; (3) At room temperature; (4) At heat temperature; (5) By sonocrystallization, nimesulide crystallized from ethanol as: (6) At cool temperature; (7) At room temperature; (8) At heat temperature; (9) By sonocrystallization, nimesulide crystallized from DMSO as: (10) At cool temperature; (11) At room temperature; (12) At heat temperature; (13) By sonocrystallization.

Crystal growth is the integration of the crystallizing components on a crystal. The two main mechanisms operating here are the diffusion of molecules from bulk to the crystal surface and surface integration, i.e. the incorporation of a growth unit into a lattice⁴.

Newer crystallization methods, such laser-induced crystallization, capillary crystallization and sonocrystallization, target the nucleation stage. These techniques by use of unusual reaction conditions (e.g. ultrasound and laser as a source of energy), are capable of bringing fortuitous surprises. Accelerating the stage of nucleation can have the remarkable influence on the crystallization state.

Nimesulide(N), chemically 4'-nitro-2'-phenoxy methane sulfonanilide, is a weakly acidic nonsteroidal anti-inflammatory drug. It differs from other nonsteroidal anti-inflammatory drugs (NSAIDs) in that its chemical structure contains a sulfonanilide moiety as the acidic group rather than a carboxylic group. Nimesulide shows high anti-inflammatory, antipyretic, and analgesic activities in addition to low toxicity, a moderate incidence of gastric side effects, and a high therapeutic index.⁶ It also exhibits a significant selectivity toward cyclooxygenase-2 (COX-2) versus COX-1 inhibition, which may explain the lower incidence of gastric side effects.

However, recent findings reported that N has a higher risk of hepatic toxicity when compared to other marketed NSAIDs.^7,8 Like many nonsteroidal anti-inflammatory drugs, N is very sparingly soluble in water (≈ 0.01 mg/mL).⁹The poor aqueous solubility and wettability of nimesulide gives rise to difficulties in pharmaceutical formulations for oral or parenteral delivery, which may lead to variable bioavailability. To overcome these drawbacks, increasing the aqueous solubility of nimesulide is an important goal.

At present polymorph screening is an empirical process in which solids are generated under various conditions and analyzed to determine solid form. Traditional approaches to polymorph generation are described by Guillory. These procedures include crystallization from single solvents or solvent mixtures, and non-solvent methods such as sublimation, thermal treatment, and crystallization from melt ¹⁰.

In present investigation we deal with the solid state characteristics of nimesulide. Moreover the study also determines how different recrystallization conditions effects the solid state characteristics of nimesulide and its dissolution efficiency.

Fig.2.X-ray powder diffraction patterns of nimesulide crystallized from methanol as:

(A) In cool temperature; (B) In room temperature; (C) In heat temperature; (D) By ultrasonic energy, nimesulide crystallized from ethanol as: (E) In cool temperature; (F) In room temperature; (G) In heat temperature; (H) By ultrasonic energy, nimesulide crystallized from DMSO as: (I) In cool temperature; (J) In room temperature; (K) In heat temperature; (L) By ultrasonic energy.

MATERIALS AND METHODS:

Materials:

Nimesulide was obtained as a pure drug from the Glenmark pharmaceuticals Nashik, India. All the solvents used were of analytical grades and obtained from the Universal Laboratories, Mumbai, India.

Preparation of nimesulide Polymorphs:

Recrystallization:

Methanol, Ethanol and DMSO were the three solvents used to study effect of solvent on development of crystal habit in changed environment. All the three solvents were saturated with the product holding the temperature slightly below the boiling point. The hot suspension was then rapidly filtered through the Whatman filter paper at room temperature. The filtered solution was then subjected to different recrystallization conditions such as:

1) Room temperature (25-30˚C).

2) Cool temperature (3-4˚C).

3) Heat temperature (65-70˚C).

4) By Ultrasound. Ultrasound also known as sonocrystallization utilizes the ultrasound power characterized by a frequency of 20-100 kHz for inducing crystallization.

Evaluation of polymorphs:

Fourier transforms infrared spectroscopy (FT-IR):

The Fourier-transformed infrared (FTIR) spectra of samples were obtained, after appropriate background subtraction, using an FTIR spectrometer (Shimadzu 8400 S, Japan) equipped with a deuterated triglycine sulfate (DTGS) detector, diffuse reflectance accessory and a data station. About1-2mg of the sample was mixed with dry potassium bromide and the sample was scanned from 400 and 4000 cm^-1.

X-ray powder diffraction studies (XRD):

Samples were prepared by pulverizing in a mortar. The XRPD patterns of samples were recorded by using a Philips PW 1830 X-ray diffractometer. Samples were irradiated with monochromatized Cu Kα radiation (1.542 A°) for measuring the 2ø range 3 to 80^o with reproducibility of ± 0.001° on a diffractometer. The XRD patterns were recorded automatically using rate meter with the time constant of 2 X 10² pulses per second and the scanning speed of 2° (2θ)/min.

Differential scanning calorimetry (DSC):

Calorimetric analyses were performed with a DSC mod. TA 4000 (Mettler), equipped with a measuring cell DSC 20. The calibration of the instrument was performed with indium, zinc and lead for the temperature, and with indium for the measurement of the enthalpy. Samples, containing about 2 mg were placed in pierced aluminum pans and heated at the scanning rates of 10 and 40˚C per min from 30 to 220˚C, under air atmosphere.

Scanning electron microscopy analyses (SEM):

The shape and surface characteristics of the samples were observed by SEM. The samples were coated with thin gold–palladium layer by sputter coater unit (VG Microtech, UK) and examined using a JEOL-840 Scanning Electron Microscopy operating with an acceleration voltage of 10 kV using the secondary electron technique.

RESULT AND DISCUSSION:

Fig. 1 shows the scanning electron micrographs (SEM) of untreated and recrystallized nimesulide from methanol, ethanol, DMSO under different conditions. It is clear from the figure that the untreated nimesulide crystals is rhombic, whereas the crystals obtained from methanol and ethanol using different conditions are needle shape and DMSO yields irregular type or rod shape crystals. While recrystallized using sonocrystallization, the shape of crystals unchanged but it affects size of crystals.

The results also showed that the size of crystals produced from methanol, ethanol and DMSO under various conditions, is significantly different from the size of untreated nimesulide. Size of crystals form at cool and heat temperature are small. Therefore, it can be concluded that increasing the rate of cooling, decreased the crystal size, due to incomplete growth of a large number of small crystals. In other words, the lower temperature causes the higher supersaturation leading to the increased nucleation and many small crystals. ¹¹ Ultrasound has significant effect on reduction of the agglomeration. Three ultrasonic effects may contribute to this phenomenon. Firstly, the shock wave, which is caused by cavitations, can shorten the contact time to bond together after contact. Secondly, agglomeration always occurs at nucleation stage. The nuclei have high surface area to volume ratio, which causes high surface tension .Nuclei are prone to adhere together to decrease the surface tension. The surface tension decreases as the crystals grow larger and therefore the crystals will become stable and do not easily agglomerate.

Fig.3.DSC Thermograms of nimesulide crystallized from methanol as:

a) In cool temperature; (b) In room temperature; (c) In heat temperature; (d) By ultrasonic energy, nimesulide crystallized from ethanol as: (e) In cool temperature; (f) In room temperature; (g) In heat temperature; (h) By ultrasonic energy, nimesulide crystallized from DMSO as: (i) In cool temperature; (j) In room temperature; (k) In heat temperature; (l) By ultrasonic energy.

Comparing SEM of crystals obtained from methanol, ethanol and DMSO solutions shows that the size and shape of crystals produced in these solvents are significantly different. The variations in face dimensions or the appearance or disappearance of some faces could be the cause of the change in morphology of nimesulide crystals, obtained from different solvents. Under certain conditions of crystallization, the growth of one set of crystal faces may be retarded, or the other set of faces may be induced to grow faster. For instance, using different solvents as a crystallization medium with the same method changes the pattern of crystal growth from rhombic to needle shape (with methanol and ethanol) and to irregular rod shape (with DMSO solution). This can be explained by the interaction between the solvent molecules and different crystal faces which is believed to change the crystal morphology. It is suggested that polar solvents were preferentially adsorbed by polar faces and non-polar solvents by non-polar faces.¹² Interaction of methanol and ethanol is stronger than ethanol due to its relatively high polarizability, indicating the higher solubility of nimesulide in methanol and ethanol rather than DMSO, this variability in polarizability results in growth of crystals from different sides and thereby different crystal habits. Compared with mechanic agitation, ultrasound has a great advantage as mixing is improved. After introducing ultrasound into the crystallization process, the mixing is improved and the local nuclei population is controlled. The nucleation rate and probability of contact between nuclei is decrease. The agglomeration is also significantly suppressed. In the other experiments, when the mixing conditions are improved, the crystals can grow larger, and agglomeration can be controlled.

To gain information on the physicochemical characteristics of the prepared crystals, X-ray powder diffraction, FT-IR spectroscopic and thermo-analytical (DSC) measurements were conducted. The purpose of these studies was to evaluate possible polymorphic modification of nimesulide crystals.

XRD spectra for all nimesulide crystals are presented in Fig. 2. Disappearance of sharp peaks in diffractograms of many samples indicates polymorphic modifications. Habit modification from crystalline to amorphous form. On the other hand the intensity of peaks in some treated samples is higher than that of untreated nimesulide. This is probably due to the higher crystal perfection or different preferred orientations of the crystals in the sample holder because of their different crystal habits. Therefore the abundance of the planes exposed to the X-ray source would have been altered, producing the radiation in the relative intensities of the peak.

DSC thermograms of recrystallized crystals are shown in Fig.3. It should be noted that the DSC thermograms of all treated samples were identical. The DSC curves of recrystallized samples showed a single endothermic peak in between 132.1 to 139.2^oC corresponding to the melting of the drug. According to the results, crystallization at different temperature was slightly altered the melting point, Tm, of the crystals, but no effect on enthalpy of fusion of the crystals. This little change in DSC data may be an effect of crystal size.

Fig.4.FT-IR Spectra of nimesulide crystallized from methanol as:

(a) In cool temperature; (b) In room temperature; (c) In heat temperature; (d) By ultrasonic energy, nimesulide crystallized from ethanol as: (e) In cool temperature; (f) In room temperature; (g) In heat temperature; (h) By ultrasonic energy, nimesulide crystallized from DMSO as: (i) In cool temperature; (j) In room temperature; (k) In heat temperature; (l) By ultrasonic energy.

The spectra of all modified crystals (Fig.4) were identical and the main absorption bands of nimesulide appeared in all of the spectra. Main absorption bands of nimesulide are as 1081.99 cm^-1 is due to S=O stretching. The intense bands at 1521 cm^-1& 1342 cm^-¹are due to asymmetric and symmetric stretching vibrations of N-O band. More than one band in between 1217-1282 cm^-1 is due to asymmetric C-O stretching of diaryl ether. The methyl C-H stretching shows peaks at 2842-2929 cm^-1band of C-H aromatic stretch appears at 3089 cm^-1. The N-H stretching band appears at 3288 cm^-1.This indicates that there were no difference between the internal structure and conformations of these samples, because these were not associated with changes at molecular level and that the altered XRD spectra for these samples were not associated with changes at the molecular level.

Results from FT-IR spectroscopy, X-ray analysis and DSC taken together led to conclusion that only habit modifications were observed during recrystallization of nimesulide under various conditions of crystallization.

CONCLUSION:

Crystallization medium has a major effect on nimesulide crystal habit modification. Crystallization of nimesulide results needle shape crystals from methanol and ethanol and irregular rod shape with DMSO. Changing the crystallization temperature only altered the size of crystals. Recrystallization by Ultrasound results in a reduction in particle size.

ACKNOWLEDGEMENTS:

The authors thank Glenmark Pharmaceuticals, Nashik, India for the gift sample of Nimesulide. We are grateful to University of Pune for providing facilities of SEM and PXRD.

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Received on 21.07.2008 Modified on 22.08.2008

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008;Page 381-385