EFFECT OF CRYSTALLINE TO AMORPHOUS CONVERSIONS ON SOLUBILITY OF CEFUROXIME AXETIL

This study investigates a new method of converting a crystalline drug to its amorphous form and studying the effect this conversion has on the solubility of the drug. The physical form of the drug is of great importance in the process of taking a drug molecule from its discovery to its formulation into an acceptable dosage form. For oral solid dosage forms, the physical state of the drug molecule influences various important parameters including bioavailability and stability. Bioavailability in tum is governed by factors like dissolution and solubility, which can be altered to give desired results. A review of the various methods involved in solid state modifications is provided to enable the reader to comprehend the importance and extensive applications of these techniques in the field of preformulation and pharmaceutical dosage form development. This study uses a poorly water-soluble drug, cefuroxime axetil, and determines the effects the treatment processes have on its solubility. The processes differ in that one renders the drug totally amorphous while the other disrupts the crystalline structure to an extent where it is in a partially crystalline state. The varied purposes of polymers used in this study have also been investigated. Polymers have been used to enhance solubility, act as impurities in the crystal structure, and in solution as crystallization inhibitors. The results of this study show that appropriate processpolymer combinations can yield significant results for solubility enhancements.

( ACKNOWLEDGEMENTS I take this opportunity to express my sincere gratitude to my major advisor, Dr.
Hossein Zia, whose inexhuastible energy has been a source of inspiration for me, throughout my years at URI. His support and efforts have made, what otherwise appeared to be a daunting task, an exciting process of learning. I thank him for being there whenever I needed him, for patiently answering every query, and for being that source of motivation I so earnestly needed. I have also to thank Dr.
Thomas Needham, whose guidance through this entire project has been a valuable asset. His ideas and suggestions have helped refine the project and are an indispensible part of this work. I would also like to thank Dr. Chong Lee for his invaluable guidance, right from his course, through my project. Thank you for being a part of my committee. My sincere gratitude to Dr. Keykavous Parang for serving as Chair on my committee.
It is a pleasure for me to be able to thank my family-dad, mom, yusra, munnu and wadood, without your support I would not have been able to see myself as I do today. Daddy and Mummy -thank you for believing in me and for giving me this opportunity, that has helped me to learn and understand the value of every little thing you did for me. To Yusra -my sister and best friend, we are miles apart and yet so close. Some of my most cherished moments here were those that I shared with you, those hours long sisterly talk that we had and those advices that we always give each other. Munazzah and Wadood -the life of our family. Your crazy antics have always put a smile on our face and pulled us through some of the toughest times together. I want you all to know that I have always loved you and missed you every day that I have been here.
I would like to thank all my friends, Dipti, Rina, Sejal, Keyur, Niraj, Chandra, Vikas, Sekhar and Gautam, for making my stay here, like home away from home.
Dipti-Keyur, Rina-Sekhar -I will always feel fortunate to have friends like you.
Among other things, staying with you guys I learned to cook, which at one point in time, was unimaginable ! ! ! Niraj -I don't know from where our friendship started, but it is something I will cherish for a long time to come. You helped me all those times when I didn't know what to do or what to say (and those were a lot of times). I owe you big time. Chandra, I have to thank you not only for helping me with my research, but for being a good friend. Your every honest opinion is appreciated. Vikas, thank you for providing those moments of relief that were so needed. I've enjoyed all the times we've spent together and all the smiles we've shared together. And then there was the time when I thought I would be all alone, and lo and behold -there was Sejal and Gautam. You've seen me through some of the hardest times, and all I can say is thank you. To all my friends -I enjoyed the times we spent together, the laughter and the tears we shared, the crazy trips that we planned together, and all the lovely food you guys treated me to!!!! Thank you for being there.

Solid States
The existence of drugs and excipients m multiple physical forms (e.g. polymorphs, isomers) provides pharmaceutical scientists with an opportunity to select the preferred form(s) of the materials to be used in a formulation. This is very useful since critical properties, such as particle morphology and solubility, frequently vary between the different physical forms of a given active pharmaceutical ingredient (API) 1 • The amorphous form of pharmacologically active materials has received considerable attention because, in theory, this form represents the most energetic solid state of a material, and thus it should provide the biggest advantage in terms of solubility and bioavailability2. Differences in the physical properties of various solid forms have an important effect on the processing of API's into drug products 3 , while differences in solubility may have implications on the absorption of the active drug from its dosage form 4 , by affecting dissolution rate and possibly the mass transport of the molecules.
The most significant physical property of the solid state is the high degree of order in which substances (e.g. metals, minerals) usually exist. The structure may be crystalline and lattice-like or non-crystalline, such as in plastic, glass or gels, which are not lattice-like or only partly so. These latter materials do have, however, much more order than liquids and gases. These materials also have, in varying degrees, some plastic and elastic properties wherein some resistance to applied stresses exists, but when the stress reaches certain intensity, either flow or fracture ensues. Although different classifications exist, four major types of ( bonds hold solids together; the strong bonds impart higher melting points to drug substances. In order of decreasing strength, the bond types are metallic, ionic (salts), valence (diamond) and molecular (many organic compounds). Thus, in some solids, the atoms or molecules or ions may be arranged in a regularly repeating pattern (crystalline state), whereas other solids are considered noncrystalline or amorphous if they do not have this characteristic of regularity5.

Characteristics and Significance of the Crystalline and Amorphous States
During the final stage of developing a new drug entity, a great deal of emphasis is placed on obtaining material of high purity and reproducibility in terms of its physical, chemical, and biological properties. Every effort is made to ensure a high degree of crystallinity, wherein the molecules have regular and well-defined molecular packing, and emphasis is also placed on whether or not the compound can exist in polymorphic or solvated crystal forms 6 . These forms can have different thermodynamic properties (e.g., melting temperature, vapor pressure, solubility), and knowledge of their existence is required to anticipate spontaneous changes in the properties of the solid during storage and/or handling of the material. It is also possible that upon isolation the material will be obtained in a fully or partially amorphous state 7 . Many compounds, especially complex organic molecules, will naturally occur or are capable of being manipulated to exist in more than one form as solids. Some of these forms are crystalline phases while others are meta-stable states where the compound is in a noncrystalline or molecularly dispersed form. Pharmaceutical scientists have been making use of the differences in the physical chemical properties that exist between these various solid states to optimize drug delivery 8 .

Crystalline Solids
The crystalline state is thermodynamically favored for solids. It is characterized by the three-dimensional order of the molecules within its crystal lattice. The molecules are arranged in a pattern that minimizes the total energy of the crystal, both kinetic and potential. This usually results in a minimum amount of void space between the molecules, as there is a maximization of intermolecular contact (bonding). The packing arrangement for a compound will be a function of both its molecular shape and the chemical groups in the molecule involved in directional intermolecular bonding, e.g., hydrogen bonding, charge transfer interaction, and dipole-dipole interactions 8

Amorphous Solids
The three-dimensional long-range order that normally exists m a crystalline material does not exist in the amorphous state, and the position of molecules relative to one another is more random as in the liquid state 2 . These materials are referred to as glasses, noncrystalline, amorphous, and vitreous solids. Typically amorphous solids exhibit short-range order over a few molecular dimensions and have physical properties quite different from those of their corresponding crystalline states. The noncrystalline state is thermodynamically unstable and there will be a tendency to entropically drive these solids to a stable crystalline state. However, since the diffusional process is slow in the solid state, it is in many instances possible to isolate and make use of these materials in dosage forms . The free energy of these noncrystalline solids is much higher than that of their corresponding crystalline forms. As a result of its higher internal energy, the amorphous state should have enhanced thermodynamic properties relative to the crystalline state (e.g. , solubility, vapor pressure) and greater molecular motion.
The high internal energy and specific volume can lead to enhanced dissolution and bioavailability 10 , but can also create the possibility that during processing or , and (iv) the prevention of chemical degradation and microbial growth through antioxidant, pH buffer, preservatives, etc.

Solubilization by Solid-State Modification
The solubility behavior of drugs remains one of the most challenging aspects in formulation development. With the advent of combinatorial chemistry and high ( throughput screerung, the number of poorly water-soluble compounds has dramatically increased and the formulation of poorly soluble compounds for oral delivery now presents one of the most frequent and greatest challenges to formulation scientists in the pharmaceutical industry2 3 .
Together with permeability, the solubility behavior of a drug is a key determinant of its oral bioavailability. Consideration of the modified Noyes-Whitney equation 24 provides some hints as to how the dissolution rate of even very poorly soluble compounds might be improved so that the limitations to oral availability can be minimized: where, dC/dt is the rate of dissolution, A is the surface area available for dissolution, D is the diffusion coefficient of the compound, Cs is the solubility of the compound in the dissolution medium, C is the concentration of the drug in the medium at time t and his the thickness of the diffusion boundary layer adjacent to the surface of the dissolving compound. The main possibilities for improving dissolution according to this analysis are to increase the surface area available for dissolution by decreasing the particle size of the solid compound and/or by optimizing the wetting characteristics of the compound surface, to decrease the boundary layer thickness, to ensure sink conditions for dissolution and last but not the least, to improve the apparent solubility of the drug under physiologically relevant conditions. Of these possibilities, the most attractive option for increasing the release rate is the improvement of the solubility through formulation approaches. Table 1 summanzes the vanous formulation and chemical approaches that can be taken to improve the solubility or to increase the available surface area for dissolution.
Of the physical approaches, the use of polymorphs 25 , the amorphous form of the drug 2 , and complexation 26 ' 27 have been widely reviewed. Decreasing the particle size of the compound by milling the drug powder theoretically results in an increase in the available area for dissolution, but in some cases the micronized powder tends to agglomerate, thereby at least partly negating the milling procedure. Presenting the compound as a molecular dispersion, on the other hand, combines the benefits of a local increase in the solubility and maximizing the surface area of the compound that comes in contact with the dissolution medium as the carrier dissolves.

Crystal Defects
The crystal lattice is generally regarded as a highly ordered structure that repeats itself in three dimensions. Crystal defects can broadly be classified into point and lattice defects (see Figure   1 )  Even in a crystal of 99.9% purity, one molecule in ten in any given direction is likely to be an impurity molecule 40 , perturbing the crystalline order of the lattice and increasing the lattice strain. As a result of the incorporation of an impurity, the increase in enthalpy of the solid itself due to the increased lattice strain may be partially offset by an increase in entropy, corresponding to the accompanying disorder, so that the corresponding increase in Gibbs free energy may not be large. However, because equilibrium properties such as solubility and solid

Solid Dispersions and Solid Solutions
In 1961, a unique approach of solid dispersion to reduce the particle size and Solid solutions containing a poorly water soluble drug dissolved in a carrier with relatively good aqueous solubility achieve a faster dissolution rate than a eutectic mixture because the particle size of the drug in the solid solution is reduced to a minimum state, i.e., its molecular size, and the dissolution rate is determined by the dissolution rate of the carrier 57 • By judicious selection of a carrier, the dissolution rate of the drug can be increased by up to several orders of magnitude.
In addition to the reduction of the crystalline size, the following factors may contribute to the faster dissolution rate of a drug dispersed in these systems 54 , a) An increase in drug solubility may occur if the majority of its solid crystallites are extreme I y small 9.
( b) A solubilization effect by the carrier may operate in the microenvironrnent (diffusion layer) immediately surrouding the drug particle in the early stages of dissolution since the carrier completely dissolves in a short time.
This was demonstrated by the faster dissolution rate of acetaminophen from its physical mixture with urea than that of the pure compound with comparable particle size 58 .
c) The absence of aggregation and agglomeration between fine crystallites of the pure hydrophobic drug may play a far more important role in increasing rates of dissolution and absorption than is presently recognized.
Serious drawbacks of aggregation and agglomeration and lumping in the dissolution medium between pure drug particles are, however, rarely present in most solid dispersion systems because the individually dispersed particles are surrounded in a matrix of carrier particles. It must be emphasized that the aggregation and agglomeration of the solid dispersion powders may not significantly affect the dissolution of the drug, which can still disintegrate quickly due to the more rapid dissolution of the soluble carrier. This advantage of solid dispersion systems was demonstrated in the in vivo absorption 59 of griseofulvin when dispersed in polyethylene glycol 6000 (10% w/w) and compressed into a hard tablet.
The dissolution rate of the dispersed drug was found to be 25 times that of the pure drug. d) Excellent wettability and dispersibility of a drug from these systems, prepared with a water-soluble matrix result in an increased dissolution rate of the drug in aqueous media. This is due to the fact that each single crystallite of the drug is very intimately encircled by the soluble carrier which can readily dissolve and cause the water to contact and wet the drug particle. As a consequence, a fine homogenous suspension of a drug can be easily obtained with minimum stirring 52 .
The methods that have been used to characterize solid dispersions are summarized in Table 2. Among these, the most important methods are thermoanalytical, Xray diffraction, infrared spectroscopy and measurement of the release rate of the drug. In addition to characterizing the solid dispersion, these methods can be used to differentiate between solid solutions (molecularly dispersed drug), solid dispersions in which the drug is only partly molecularly dispersed and physical mixtures of the drug and carrier2 3 .

Polymorphic Transformations
Many drug substances can exist in more than one crystalline form with different space lattice arrangements. This property is known as polymorphism. The different crystal forms are called polymorphs. Crystalline polymorphs have the same chemical composition but different internal crystal structures and, therefore, possess different physicochemical properties. The different crystal structures in polymorphs arise when the drug substance crystallizes in different crystal packing arrangements and/or different conformations. The occurrence of polymorphism is quite common among organic molecules, and a large number of polymorphic drug ( Dissolution testing Thermoanalytical methods: differential thermoanalysis and hot stage rrucroscopy Calorimetric analysis of the solution or melting enthalpy for calculation of entropy change X-Ray diffraction Spectroscopic methods, e.g. IR spectroscopy Microscopic methods including polarization microscopy and scanning electron microscopy In such cases, a more soluble and faster dissolving form may be utilized to improve the rate and extent of bioavailability.

Solid-State Characterization
Many methods are available that can contribute information regarding the nature of a solid system. In many instances, a combination of two or more methods is required to study its complete picture. The analytical techniques used in this study will be discussed briefly in the following section.

X-Ray Diffraction
In 1912, Max Von Laue pointed out that if the wavelength of electromagnetic radiation became as small as the distance between atoms in the crystals, a diffraction pattern should result. Later it was found that the X-ray region has the right wavelength and a definite diffraction pattern was obtained for copper sulfate ( crystals. In essence, the crystal diffracts X-rays similar to a diffraction grating, whose plane diffracts ordinary light. The three-dimensional crystal functions like a series of plane gratings stacked one above the other 5 . For a single crystal the diffracted X-rays consist of a few lines; with powder, due to a random distribution of crystals, the diffraction pattern consists of a series of concentric cones with a common apex on the sample. The atoms in a crystal possess the power of diffracting the X-ray beam. Each substance scatters the beam in a particular diffracting pattern, producing a fingerprint for each atomic crystal or molecule.
Powder X-ray diffraction analysis is employed for characterization of crystalline structure. It has been used to determine the existence of polymoprhic forms of many substances. It is a very important and efficient tool in studying the physical nature of solids.

Differential Scanning Calorimetry (DSC)
Thermal analysis is a technique in which a physical property of a substance is monitored as a function of controlled temperature increase. Modern thermal analytical methods can measure weight loss on heating, melting points, heat and energy of transitions and changes in form, in dimensions or in the viscoelastic properties of the substance. They find wide application in material characterization, purity of medicinal substances, study of relative heat stabilities and dynamic properties of new compounds, as well as in crystallography, chemical kinetics and generation of phase diagrams 5 . Most thermodynamic events are accompanied by a loss of heat or require addition of heat from an ( external source in order to proceed. Each of these occurrences can be followed thermodynamically by noting either change of temperature of the sample under study or energy changes of the sample with respect to time. Thermal analyses include thermogravimetry (thermogravimetric analysis, TGA), differential thermal analysis (DT A) and differential scanning calorimetry (DSC). DSC is very closely related to TGA, but differs only in that the sample and reference containers are not contiguous, but are heated separately by individual coils that are heated (or cooled) at the same rate. Platinum resistance thermometers monitor the temperature of the sample and reference holders and electronically maintain the temperature of the two holders constant. If a thermodynamic event occurs which is either endothermic or exothermic, the power requirements for the coils maintaining a constant temperature will differ. This power difference is plotted as a function of the temperature recorded by the programming device. Unlike DT A, in DSC the amount of heat put into the system is exactly equivalent to the amount of heat absorbed or liberated during a specifc transition (transition energy).

Optical Microscopy
The past decade has witnessed an enormous growth in the application of optical microscopy for micron and sub-micron level investigations in a wide variety of disciplines. Microscopy has been used quite often to study the morphology of solids, alone, in mixtures or dispersions, and in polymorphism. The properties of the particles in a powder can influence profoundly the pharmaceutical performance of the solid, such as the processmg, compaction, stability, and/or release rate of the active constituent.
Optical microscopy can be used to study the crystal habit (shape and roughness) of crystalline substances.
The physical characterization of amorphous solids utilizes a wide range of techniques and offers several types of

Cefuroxime Axetil
Cefuroxime axetil (CA), an acetoxyethyl ester prodrug of cefuroxime, was the first oral cephalosporin of the second generation to be commercially available as   drug, resulting in high urinary concentrations of cefuroxime. Cefuroxime axetil is associated with a low incidence of adverse events, with gastrointestinal disturbances being the most frequently observed. Thus it is an effective and convenient treatment for a wide range of infections, and may be considered a therapeutic option when empirical treatment of community-acquired infections is required.

Objectives of the Study
The crystalline nature of a drug has a direct effect on its solubility, and may be a limiting factor for its dissolution and bioavailability. A reduction in the crystallinity of the drug or its conversion to the zero -crystalline (amorphous) state, would be a means of enhancing the bioavailability of the drug. However, the unstable nature of the amorphous state, accelerated by external factors (e.g. moisture), limits its wider and more frequent application.
This study primarily investigates the effect of crystal modification on the solubility of CA and its individual isomers. Techniques used have been employed with the aim of altering the crystal structure. A comparative study between the processes has also been used to determine the varied effect on the solid-state of the drug. Polymers have also been used, in conjunction with the processes; to investigate any positive effect on the ability of the drug to retain its modified state (of improved solubility). Use of polymers has been considered as they not only assist in improving solubility; but can also act as impurity (in small quantities), to destabilize the rigid crystal structure. Since the drug exists as a mixture of ( diastereoisomers, we can expect a difference in the effects of the processes and polymers on the individual isomers. Diastereoisomers have different solubilities and melting points, and hence will show differences in the degree of conversion from crystalline to partially crystalline to amorphous states.

SECTION II: EXPERIMENTAL
The solid state of a drug affects its solubility, and in tum its dissolution and bioavailability from the dosage form. The existence of the drug in the crystalline or amorphous form needs to be adequately characterized. Crystalline to partially crystalline or amorphous conversions have been studied in this project. The primary aim of this study was to determine the effect of processes and polymers on the crystalline to amorphous conversions and their effect on the aqueous solubility of the drug. Solubility is one of the parameters used to monitor these conversions. In addition, analytical techniques like DSC (Differential Scanning Calorimetry), XRD (X-Ray Diffraction), and optical microscopy were used to characterize these conversions.

Equipments I Reagents:
Equipments

I. 2 Standard Procedure:
1) Mobile phase used m this study is a mixture of 0.2 M monobasic ammonium phosphate and methanol (620:380).
2) Stock solution (1 mg/ml) of cefuroxime axetil in methanol is used for the preparation of the standard solutions.
4) The HPLC system was prepared by setting the following parameters, a) The column thermostat temperature should be set to 32 °C.
b) The absorbance of the spectrophotometer should be set to 278 nm.
c) The flow rate should be set to 1 ml/minute.

5)
The assay for the USP reference standard and the sample of cefuroxime axetil were identical, performed using the standard procedure above.
6) The calibration curve is constructed by plotting the area under the curve (AUC) against their concentrations.

7)
Since the drug exists as a mixture of diastereoisomers, the total concentration 1s given by the sum total of the concentration of the individual isomers.

8) Separate calibration curves for the individual isomers as well as the total
drug have been constructed, which can be used to determine the unknown concentration of the samples. 9) Validation of the assay for cefuroxime axetil was performed over five days to ensure reproducibility.
The drug exists as a mixture of diastereoisomers A and B that elute separately.
Isomer B (-10.5 min) elutes first followed by isomer A (-11.5 min) with a difference of approximately 1 minute between them (see Figure 5).    (   Table 4 shows the data for the calibration curve of cefuroxime axetil. Tables 5   and 6 show the data in terms of the individual isomers A and B. Table 7 shows the percentage of isomers A and B in the cefuroxime axetil sample, and it can be seen that the ratio of the isomers lie within the USP limits. Figures 8 and 9 illustrate the linear portion of the calibration curve for the sample and its individual isomers, respectively. The correlation coefficient was found to be 0.9995 and 1.0 for cefuroxime axetil (USP) and the cefuroxime axetil sample respectively. Table 8 shows the mean values of the data generated out of five days of replication studies for seven separate and distinct samples for series of cefuroxime axetil concentrations ranging from 10 to 200 µg/ml.
The purity of the sample of cefuroxime axetil was determined as shown in Table   9. The average percentage purity was found to be 103.85% with a standard deviation of 4.462, when compared to the USP reference standard.

Solubility Studies of Cefuroxime Axetil
Solubility studies of the drug were carried out in water and 0.07 N HCl, for a period of 24 hours with samples withdrawn and analyzed at seven time intervals.
The 24-hour time duration was selected to allow the drug to reach its equilibrium solubility. The USP dissolution test for cefuroxime axetil tablets uses 0.07 N HCl as the dissolution medium in order to simulate gastric conditions; hence it has been selected as one of the media for solubility studies.         Table 9: Determination of the percentage purity of the cefuroxime axetil sample based on the USP reference standard As observed from the solubility profile of the drug (Figure 10), CA reaches its equilibrium solubility by 4 hours and remains at equilibrium for over 10 hours.
An excess amount of the drug in the medium ensured equilibrium and also accounted for any degradation that may have taken place during the 24 hour time period. The studies were conducted at room temperature (25 °C The solubility of the USP reference standard was determined in water and 0.07 N HCl (see Figure 11). The untreated cefuroxime axetil sample was first tested to determine its solubility profile. The drug was then treated with a combination of processes and polymers. The solubility of these processed mixtures was then determined by the above procedure. The purity of the drug after treatment (with ( process and/or polymer), was also determined to rule out any interactions /loss /degradation of the drug.

Modification of Physical State
This section deals with the various processes and polymers that were employed to bring about a modification in the solid state of the drug. Each factor is dealt with individually in a separate sub-section.

Selection of Polymers
The use of a polymer serves three purposes; enhances solubility, acts as an impurity to destabilize the rigid crystal structure 2 , and in the amorphous state, assists in preventing the re-conversion of the unstable amorphous state to its stable crystalline state 5 . This approach was adopted as the unstable nature of the amorphous state was recognized and known to limit its wider application.

Selection of Processes
In this study, the process of microfluidization has been used to assist m converting the drug from its crystalline to amorphous form. Microfluidization is a widely used technique for the homogenization of emulsions and dispersions, liposomes and cell disruption. The M-11 OS Microfluidizer® Processor (Microfluidics Corporation, Newton, MA) shown in Figure 12, contains an airpowered intensifier pump designed to supply the desired pressure at a constant level to the product stream 10 • As the pump travels through its pressure stroke, it drives the product at a constant pressure through precisely defined fixedgeometry microchannels within the interaction chamber ( Figure 13). As a result, the product stream accelerates to high velocities, creating shear rates within the product streams that are orders of magnitude greater than any other conventional means. All of the product experiences identical processing conditions, producing the desired results, including uniform particle size, and droplet size reduction (often submicron), deagglomeration, and high yield cell disruption. This study uses an existing technique for a new application, the modification of the crystal structure. A solution of the drug and/or polymer in an appropriate solvent system was prepared, and then passed through the microfluidizer under a defined set of conditions. The solvents were removed, and the treated drug characterized by solubility, DSC, XRD and microscopy. A system of drug/polymer in similar solvents was also prepared by solvent evaporation to study effects of solvent, if any. A physical mixture of the drug and polymer was also studied to determine any effects of polymer in the absence This physical mixture of the drug and polymer was then used for solubility determinations and other characterizations. Table 10 lists all the drug-polymer-process combinations that have been studied in this project.

DSC Studies
DSC analysis was carried out usmg Perkin Elmer DSC7 (Perkin Elmer, Wellesley, MA). Samples were weighed (approximately 10 mg), sealed in aluminum pans and loaded into the pan holder. An empty pan was used as blank. The scanning rate was set to 10 °C/min. Three separate samples were tested for the drug and each of the processed mixtures.

Results and Discussion
The solubility profiles of the USP reference standard ( Figure 11) and the sample ( Figure 10) were determined. The USP reference standard was found to be in the amorphous form, as confirmed by the XRD data which showed an absence  ." of peaks (see Figure 14). Over the duration of the solubility experiments, the amorphous starting material begins converting to its stable crystalline form, as evident from the solubility profile of the USP reference standard (Figure 11 ).
The commercial sample exhibited multiple peaks in the XRD (Figure 15) indicating the presence of the crystalline form . The DSC data for the commercial sample ( Figure 16) shows the presence of two melting endotherms, at 123 °C and at 173 °C, for the two isomers B and A, respectively. The drug degrades (at -175 °C) after the melting of the second isomer, as observed from the loss of the straight nature of the baseline. Also, visual examination shows browning and charring of the drug after -176 °C when examined on the melting point apparatus. The drug in the solid form exists as a 1: 1 mixture of the two isomers, A and B. However, in solution, isomer B is in greater concentration than isomer A due to its significantly greater solubility. In aqueous solution, the ratio of the solubilized isomers is 9: 1 (B:A) (see Figure 17). This is also true for the solubility determinations made in 0.07 N HCl. The solubility of isomer B is -5 times the solubility of isomer A ( Figure 18).
Solubility determinations, using excess amounts of drug, follow a zero-order kinetics, in which the concentration in solution depends on the drug's solubility.
Excess drug replaces any drug lost due to degradation in solution, whereby more drug is released from the suspended particles so that the amount of dissolved drug remains constant. This concentration is the drug's equilibrium solubility in a particular solvent at a particular temperature 11 • It is important to note that the amount of drug in solution remains constant despite its decomposition with time. linearly. This approximates first-order kinetics in that the rate of dissolution is proportional to time. As the time increases, more and more drug goes into solution, until the solution is saturated with the drug, and zero-order or saturation kinetics is observed.
In the following section, the effect on solubility of the drug has been compared in the absence and in the presence of the selected polymers.

I Effect of crystalline to amorphous conversion on solubility of the drug in the absence of polymers
The solubility profiles of the untreated drug, its microfluidized form and its recrystallized form were examined. As seen in Figure 19 both the processes bring about an increase in the solubility of CA. However, the recrystallized drug shows a dramatic decrease in solubility after the first two hours, reducing the amount of drug dissolved to the solubility of untreated CA after 24 hours. while the endotherm for isomer B is not present. However, the heat of fusion of the recrystallized isomer A is lower than that of the pure isomer A (Table 11 ). 11.55 CA + PVP (recrystallized) 8.67 CA+ Eudragit RD100 (recrystallized) 10.78 Table 11: Heat of fusion (J/g) of isomer A for the different process-polymer combinations (the remaining process-polymer combinations convert the isomers to its amorphous form) ( amorphous form while the recrystallized drug is in a partially crystalline state. This is also confirmed by the comparison of the XRD data shown in Figure 21. The solubility and DSC data indicate that the higher solubility, lower melting isomer (B) is affected by both the processes and is converted to its amorphous form. In the case of the recrystallized sample, isomer (B) loses its amorphous form on prolonged exposure to the medium, as indicated by the downward slope of the solubility curve ( Figure 19). However, the microfluidized drug shows only a gradual decrease in the solubility indicating a greater resistance to conversion to its original form . The use of microfluidization for these conversions has not been studied previously and is a new phenomenon. The drug is subjected to high pressure and velocities causing complete disruption of the crystal structure. Pharmaceutical solids, as we know them, rarely exist as   Table   12 shows the purity of the physical mixtures that were found to be very close to or equal to (between 99-101 %) the pure drug. Thus, we can safely conclude that    The X-ray diffraction data for the recrystallized -PVP, Eudragit EPO, and Eudragit RDlOO samples are shown in Figure 33.    Table 11). Heat of fusion (.6.S f = .6.Hf I Tm) has been used as an indication of disruption or disorder induced by additives or impurities 16 • The X-ray diffraction data for the microfluidized -PVP, Eudragit EPO, and Eudragit RDlOO samples are shown in Figure 34. All the samples subjected to microfluidization are converted to the totally amorphous form as seen from the XRD scans and confirmed from the DSC data, indicating the absence of both the melting endotherms.
The samples prepared with Eudragit EPO show the lowest solubility, which could be attributed to the formation of the complex between the cationic polymer and the anionic drug, detected on the chromatogram as a third peak (at -12.5 min). The recrystallization process and polymer combinations involving PVP and Eudragit RD 100 do not render the drug amorphous, but a high degree of crystal disruption may be the cause of the enhanced solubility. We do notice, however, that one of the isomers is converted to the amorphous form while the other is in a partially crystalline state. The heat of fusion of the partially crystalline isomer (A) is also reduced, as seen in Table 11 . A microscopic analysis of all the processed mixtures shows a loss of the needle shaped crystalline structure of the samples, similar to the ones shown before (Figures 22 and 23). Table 14 lists the yields for the two processes along with the polymers. It was noticed that the process of microfluidization yielded very low quantities of the sample (~ 70%), whereas the recrystallization process gave significantly higher yields. Based on the statistical analysis along with the process yields, the recrystallized CA-PVP combination was selected for the preparation of the tablets.

Statistical Analysis
A two-factor factorial design is used to study the effect of the different processes and polymers on the solubility of the drug. In this study, we have a 3 x 4 factorial design, that is, we have 2 factors, one with 3 levels and the other with 4 levels. The two factors are polymer (C 1 ) and process (C 2 ), with 4 and 3 levels, respectively. The two independent variables, their levels and their values are summarized in Table 15. Therefore the number of treatment groups we have is 3 x 4 = 12 groups (Table 16). Data for each representative group was generated in triplicates, hence we have 36 observations, as listed in Table 17
From the table we can see that the P-values for the polymer, process, and the polymer-process interactions are lower than the a-value (0.05). Hence we can conclude that these three factors have a significant effect on the solubility of the drug. The main effects plot for process shown in Figure 35 indicates that both the processes (microfluidization or recrystallization) show an almost equivalent increase in solubility as seen from points 2 and 3 in the graph. From the interactions plot, Figure 36, it is observed that the combination of PVP with either of the processes (represented as line •-----•) would be a good choice for improving solubility. As seen from the graph, process 2 (microfluidization) and 3 (recrystallization) show a significantly higher solubility, thus making them a good choice for use in the preparation of tablets. The normal probability plot ( Figure 37) follows an almost straight line, and hence the distribution is normal and the model used is adequate. 0\ ,

Preparation of Tablets (or compacts):
Tablets of cefuroxime axetil were prepared containing an equivalent of 250 mg of cefuroxime. The total weight of the tablet was fixed at 4 70 mg (based on the marketed formulation). In addition to the drug, the tablets contained the inactive ingredients colloidal silicon dioxide, croscarmellose sodium (Ac-Di-Sol), microcrystalline cellulose (A vicel), hydroxypropyl methylcellulose, and sodium lauryl sulfate. Tablets were prepared with the untreated drug as well as the recrystallized drug-PVP mixture. In the latter formulation HPMC was omitted due to the presence of PVP. All the powders were sieved through a sieve of mesh size 40. The components of the formulation were mixed according to the formulation (Table 19) for 15 minutes on the rotary shaker. Tablets were prepared by direct compaction on the Carver press at a compression pressure of 1000 lbs. A batch of 20 tablets was prepared for each of the formulations.

Dissolution studies:
USP apparatus 2 was used. The apparatus was set to 55 rpm. The temperature of the water bath was set to 37 ± 0.5 °C. Dissolution medium used was 0.07 N HCI. For the dissolution studies, 900 ml of the dissolution medium was filled in the dissolution flasks and the system was allowed to equilibrate for 30 minutes.   5,15,30,45,60,90 and 120 minutes. At each time interval, 1 ml of the sample was withdrawn from the dissolution flask and replaced by equivalent amount of fresh dissolution medium at the same temperature. The samples were filtered by centrifugation, 0.5 ml of the filtrate was diluted with 0.5 ml of the mobile phase and the samples were analyzed by HPLC.

Results and Discussion:
The tablets prepared were tested for their drug content and their dissolution profiles were determined based on the above method. The USP limits for content uniformity are as follows, "Cefuroxime axetil tablets should contain the equivalent of not less than 90.0 percent and not more 110.0 percent of the labeled amount of cefuroxime". Six (6) tablets were used to determine the amount of drug present in the tablets. The results for the drug content in the tablets are tabulated in Table 20. As the results indicate, Formulation A contains an average of 97% and Formulation B contains an average of 99% of cefuroxime. Hence, both the formulations lie within the USP limits for drug content. The limits for the USP content uniformity have been used as a basis for determining the amount of drug in both the formulations. The dissolution profiles of the tablets prepared from the untreated drug and the drug-PVP mixture are shown in Figure 38. For dissolution, the USP limits state that "not less than 60% of the labeled amount of cefuroxime is dissolved in 15

Summary:
The modification of a crystalline drug to its amorphous or partially crystalline form has a significant effect on the solubility of the drug, as evident from this study. The new application of microfluidization, as a means of modifying the crystal structure, has been successfully evaluated. However, studies with other   The results of this investigation can be summarized as follows: 1. Microfluidization can be effectively used as a means of converting a crystalline drug to its amorphous form.
2. Polymers used in this study efficiently serve the purposes they were employed for, namely, assisting in improving the solubility of the drug, acting as impurities to destabilize the rigid crystal structure, and as crystallization inhibitors, preventing reconversion of the amorphous drug to its crystalline form .
3. Different process-polymer combinations show different effects on the individual isomers of the drug.
4. PVP was the better polymer among the three polymers used in this study.
5. Tablets/compacts prepared from the drug-polymer mixture exhibit dissolution profiles in accordance to the USP limits.