Increasing Copper Indomethacin Solubility by Coprecipitation with

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Increasing Copper Indomethacin Solubility by Coprecipitation with Poly(vinylpyrrolidone) Using the Aerosol Solvent Extraction System Louise A. Meure, Barry Warwick, Fariba Dehghani, Hubert L. Regtop, and Neil R. Foster* School of Chemical Engineering and Industrial Chemistry, The University of New South Wales, Sydney, New South Wales, Australia 2052

Copper indomethacin (Cu-Indo) is a nonsteroidal anti-inflammatory drug currently available for veterinary use. Its application is limited to oral formulations because of its poor solubility in biocompatible solvents. In this study, Cu-Indo has been coprecipitated with poly(vinylpyrrolidone) (PVP) to increase the solubility of Cu-Indo in biocompatible solvents. The aerosol solvent extraction system was used to produce Cu-Indo/PVP coprecipitates in various ratios. Carbon dioxide was used as an antisolvent to precipitate PVP and Cu-Indo from dimethylformamide solutions. Microspheres of PVP and Cu-Indo were formed that ranged in size from 50 nm to 4 µm at most conditions studied. A coprecipitate containing 10 wt % Cu-Indo and 90 wt % PVP was found to be at least 93 times more soluble in ethanol than factory-grade Cu-Indo. The significance of these results is that there may now be the potential for Cu-Indo to be used in parenteral applications. Introduction Copper indomethacin (Cu-Indo) is a nonsteroidal anti-inflammatory drug that is currently available in Australia for veterinary use. The structure of Cu-Indo can be found in previous studies by Weder et al.1,2 and Warwick et al.3 The administration of Cu-Indo is limited to oral formulations because of its poor solubility in solvents suitable for therapeutic use. For a drug to be used in a parenteral or ophthalmic application, it must have a suitable solubility in a biocompatible solvent. Parenteral administration allows fast therapeutic response and may enhance the therapeutic effect of the drug. The objective of this study was to increase the solubility of Cu-Indo in biocompatible solvents to broaden the application base and improve the delivery of the drug. The solubility was improved by coprecipitation of Cu-Indo with poly(vinylpyrrolidone) (PVP). Improving the delivery of Cu-Indo is of significance because CuIndo, while currently in veterinary use, may potentially find human application in the future. The formation of drug/polymer blends is an important process in the pharmaceutical industry, particularly when the drug is not suitable for use in its pure form. Polymers have been used to help control the factors that limit drug administration. Drug/polymer blends have been used in drug delivery to protect the drug from its surroundings, manipulate drug properties, increase the drug stability and solubility, target the drug to a specific part of the body, and control drug release.4-6 Polymers have been combined with drugs by encapsulation, coprecipitation, and complex formation. For a polymer to be used in drug delivery, it must be nontoxic, often biodegradable, have appropriate interactions with the drug, and have suitable properties (solubility, molecular weight distribution, particle size, porosity). Poly(vinylpyrrolidone) is a synthetic polymer consisting of a linear 1-vinyl-2-pyrrolidone monomer. PVP is nontoxic and odorless, has no irritant effect on the skin, causes no sensitization, and is soluble in both water and * To whom correspondence should be addressed. Tel.: +61 (2)93854341.Fax: +61(2)93855966.E-mail: [email protected].

many commonly used organic solvents. These properties make PVP attractive for use in many types of formulations. The pharmaceutical industry has made use of PVP, which is suitable for oral, parenteral, and topical applications, for over 40 years. It has been used as a binder, dispersion aid, film plasticizer, crystal growth retarder, anti-irritant, stabilizer, solubility enhancer, and viscosity modifier and in controlled-release applications.7 The dissolution profile of slow-release formulations and bioavailability of poorly soluble drugs can be improved by the addition of PVP.7 Rodriguez-Espinosa et al. discussed the various mechanisms that have been proposed to explain the increased drug dissolution rate in the presence of PVP.8 It has been suggested that the increased drug dissolution is caused by the formation of a complex because of the presence of PVP as a high energy form or the formation of a coacervate. The degree of improvement in the rate of dissolution depends on the molecular weight, which affects the viscosity, adhesive properties, and rate of oral absorption. The mechanism for PVP increasing the solubility of drugs has been stated to be due to hydrogen bonding between the drug and polymer.7 Svoboda et al. explains the ability of PVP to improve drug solubility by hydrophobic bonding and an exothermic interaction between the solubilized compound and PVP.9 It was also found, by Svoboda et al., that PVP increased the activity of acronycine, an experimental antitumor drug. Pharmaceutical compounds, such as Piroxicam,10 Frusemide,11 Reserpine,12 Sulfathiazole,13 Digitoxin,14 and the anti-inflammatory CI-987 (5-{[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-methylene}-2,4-thiazolidinedione),15 have been coprecipitated with PVP by the solvent evaporation method to increase the drug dissolution rate, solubility, and absorption. The aim of this work was to provide a form of CuIndo that may be suitable for injectable applications, by coprecipitation with PVP. Several factors influence the dissolution of an active principle from a solid pharmaceutical formulation, such as solubility, particle size, and crystal habit. The bioavailability of poorly soluble

10.1021/ie030483q CCC: $27.50 © 2004 American Chemical Society Published on Web 01/13/2004

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drugs is particularly dependent on the particle size. Small particles have faster dissolution rates and therefore have higher activity and are more readily absorbed. Conventional methods for producing micronized particles involve grinding, crushing, spray drying, sublimation, and recrystallization from solution. These processes are unsuitable for many pharmaceutical applications because the heat and mechanical stresses placed on the drug can degrade the drug and alter its properties. These processes can also leave unacceptably high levels of residual solvent, and thus further purification is needed. The conventional techniques for coprecipitation and encapsulation of drugs with polymers involve dissolution of the polymer and drug in a common solvent and then evaporation of the solvent, often leaving high levels of residual solvent. Many drugs have low solubility in solvents that can be evaporated at moderate conditions, and therefore extreme conditions may be required to remove the solvent. In the case of Cu-Indo, the solvents that are most suitable for solubilization are N,Ndimethylformamide (DMF), N-methylpyrrolidone (NMP), and dimethyl sulfoxide (DMSO). All of these solvents present problems of either elevated boiling points or toxicity. The conventional methods are therefore unsuitable for Cu-Indo because of the levels of residual solvent in the coprecipitate after evaporation. Recently, dense gas techniques have been developed for coprecipitation, encapsulation, and micronization.16 A dense gas is a fluid close to or above the critical point, generally with a reduced temperature and pressure between 0.9 and 1.2. Supercritical fluids have a liquidlike density and a diffusivity and viscosity between that of a liquid and a gas. A more gaslike diffusivity indicates a greater ease of mass transfer. A dense gas will therefore diffuse into a matrix more quickly than a liquid solvent while retaining a liquidlike solvent strength for dissolving a component from the matrix. Carbon dioxide is the most widely used dense gas because it has low critical parameters (Tc ) 304.25 K and Pc ) 7.38 MPa), is inexpensive, nonflammable, nontoxic, and noncorrosive, and is readily available in large quantities. Carbon dioxide is particularly useful for pharmaceutical and other heat-sensitive applications because its critical temperature is close to ambient temperature. Carbon dioxide is relatively miscible with a variety of organic solvents and is easily recovered after processing because of its high volatility at atmospheric conditions. The dense gas micronization and purification technique used in this study is the aerosol solvent extraction system (ASES). The ASES process utilizes dense gas technology to precipitate a solute from a liquid solvent. The ASES technique has been used extensively for processing polymers and pharmaceuticals and is capable of producing micron-sized particles with low levels of residual solvent. Techniques similar to the ASES process have also been described and are referred to as precipitation by compressed antisolvent (PCA),5,17,18 solution-enhanced dispersion by supercritical fluids (SEDS),6 and the supercritical antisolvent process (SAS).19,20 The major characteristic to be noted is the supercritical antisolvent. Dense gas antisolvents have also been used in the field of explosives, coloring matter, superconductors, catalysts, and inorganic compounds.16 In the ASES process, the solute is dissolved in a solvent and added to a dense gas phase typically by a

dispersion device, such as a nozzle. The dense gas acts as an antisolvent for the solute to be micronized and a solvent for the organic liquid. The solvent is expanded when contacted with the dense gas because of rapid diffusion of the dense gas into the solvent. The rapid expansion is caused by the large surface area of the fine droplets produced by the dispersion device and the continuous addition of more antisolvent. A rise in the saturation level of the solute in the solvent occurs as the amount of solvent available to the solute is rapidly reduced. When the saturation level is exceeded, the solute rapidly precipitates as the solvent is extracted. The ASES process has many advantages when compared to conventional coprecipitation because it produces micron-sized particles with a narrow particle size distribution and minimal residual solvent.21 The efficiency is further improved by the use of the spraying device, which is able to produce jet breakup and uniformly disperse the solution as fine droplets with large surface area to improve the diffusion processes involved in the precipitation and allow precipitation over an extremely short span of time. The ASES technique can also be operated at low temperatures and is therefore suitable for heat-sensitive materials. There have been many studies carried out on the processing of polymers by dense gas antisolvent techniques, such as the ASES process, producing microspheres and fibers: poly(L-lactide),22,23 polystyrene,17,18,24-26 and poly(acrylonitrile).18 Polymers are challenging solutes to control in the ASES process because of the glass transition temperature, which is lowered in the presence of a supercritical solvent. The supercritical fluid used to extract the solvent diffuses into the polymer and acts as a diluent, lowering the glass transition temperature of the polymer.27 The degree of reduction in the glass transition temperature is dependent on the solubility of the supercritical fluid in the polymer. If the glass transition temperature is reduced below the operating temperature, agglomeration and plasticization may occur, forming a film instead of discrete particles. Previous reports on the micronization of polymers by dense gases have shown that particle agglomeration was most likely when processing amorphous polymers with low glass transition temperatures.5,27 Of particular relevance to using PVP in gas antisolvent processes is the relatively high glass transition temperature. The PVP primarily used in this study had an average molecular weight of 10 000 and a K value (intrinsic viscosity) of 16. A PVP polymer with K ) 17 has a glass transition temperature of 399 K. Polymer particle formation is dependent on the viscosity and, therefore, polymer concentration.17,28 At high polymer concentration, fiber formation instead of microsphere formation can occur.17 Fiber formation is due to precipitation of the solute before breakup of the liquid jet occurs. The particle size and morphology can also be influenced by many variables that can be manipulated in the ASES technique. The variables are temperature, dense gas pressure and density, flow rate of the antisolvent, flow rate of the solvent, solute concentration, nozzle type and diameter, type of solvent, and type of antisolvent. The effects of process parameters have been discussed by Reverchon in a paper on supercritical antisolvent techniques.29 Warwick et al. studied the synthesis, purification, and micronization of Cu-Indo using the gas antisolvent (GAS) technique3 and also micronization of Cu-Indo

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using the ASES technique.30 Dimethylformamide was used as a solvent and CO2 as an antisolvent in the processes. In the GAS technique, the dense gas antisolvent is added to the solvent to precipitate a solute, rather than the solvent being sprayed into an antisolvent phase, such as in ASES. Cu-Indo with a purity greater than 95% was produced in a single step at 298 K, by the GAS process. Warwick et al. found that the use of CO2 as an antisolvent instead of ethanol, which is used in the conventional synthesis, increased the yield of Cu-Indo. The conventional synthesis and purification process is also very slow, taking more than 24 h. It was found that the solute concentration and expansion rate controlled the particle size and morphology in the GAS process. At slow expansion rates, rhombic crystals formed, and at fast expansion rates, rhombic and bipyramidal particles formed. Also, smaller particles were formed at faster expansion rates.3 In the ASES technique, the solute concentration, solution flow rate, solvent type, and temperature influenced the particle size and morphology.30 In this study, the ASES process is used to coprecipitate Cu-Indo. Prior to coprecipitation, a study was conducted to examine the feasibility of processing PVP by the ASES process and to optimize the ASES operating parameters for micronization of PVP. This preliminary study provided the conditions at which the coprecipitation could be carried out. Experimental Section Materials. Cu-Indo [Cu2(Indo)4(DMF)2] was supplied by Biochemical Veterinary Research with a purity of 89.58 wt %. Poly(vinylpyrrolidone) was purchased from Aldrich Chemical Co. Inc., with average Mw’s of 10 000 (polydispersity, 2.62; K value, 16) and 55 000 (K value, 31). Dimethylformamide (Bacto Laboratories Pty. Ltd., HPLC grade) and carbon dioxide (BOC Gases, industrial grade) were used in the precipitation experiments. Ethanol (BDH Laboratory Supplies, HPLC grade), acetic acid (Ajax Chemicals, analytical reagent grade), acetonitrile (BDH Laboratory Supplies, HPLC grade), tetramethylammonium chloride (Aldrich Chemical Co.), indomethacin (Sigma Chemical Co., laboratory grade), and methanol (Unichrom Asia Pacific Specialty Chemicals Ltd., HPLC grade) were used in the solubility measurements. Procedure and Apparatus. A schematic diagram of the ASES experimental apparatus used for the micronization and coprecipitation study is shown in Figure 1. The precipitation chamber (G; a Jerguson sight gauge series no. 32) was submerged in a water bath (I) to maintain the apparatus at a constant temperature. The temperature of the water bath was controlled by a recirculation heater (F; Thermoline Unistat 130 WaterHeater). The solute(s) was (were) dissolved in an organic solvent and held in a solution reservoir (C). The precipitation experiments were conducted using solutions of PVP in DMF and mixtures of PVP and CuIndo in DMF. The solution was delivered to the precipitation vessel using a HPLC pump (D; Waters Associates Chromatography Pump M-6000A). Druck pressure indicators (E) monitored the pressure of both the precipitation vessel and the organic solution injected to the system. The gas antisolvent, carbon dioxide, was supplied to the system by a syringe pump (B; ISCO 260D), which

Figure 1. Schematic diagram of the ASES apparatus: (A) CO2 cylinder, (B) syringe pump, (C) solution reservoir, (D) HPLC pump, (E) pressure transducer, (F) heater, (G) precipitation vessel, (H) filter, (I) water bath, (J) solvent trap.

was used to fill the precipitation vessel to a desired pressure. The gas antisolvent flow rate was controlled by a needle valve located before the vent. Once the system reached steady state, the organic solution was sprayed into the precipitation vessel, filled with dense gas, via a nozzle (100 µm/254 µm stainless steel capillary tubing and 50 µm Peeksil tubing, each of approximately 6 in. in length). The organic solution and dense gas antisolvent were fed to the vessel cocurrently. After precipitation, the flow of the solution to the vessel was stopped, and at least 260 mL of dense gas antisolvent was used to wash the precipitate at the operating temperature and pressure to remove organic solvent residue. The precipitate was collected on a 0.5 µm filter (H). The antisolvent passed through a solvent trap (J), where any residual solvent could be collected. The effects of process parameters, such as temperature, antisolvent density (pressure), solute concentration, nozzle diameter, solvent flow rate, and PVP molecular weight, on precipitate characteristics were investigated. A moderate temperature range of 283313 K and a pressure range of 5.5-19 MPa were used. Experiments were carried out at subcritical liquid and vapor-over-liquid antisolvent conditions and at supercritical conditions. For the two-phase condition, the precipitation vessel was partially filled with liquid antisolvent in equilibrium with vapor. The solute concentration was varied from 1 to 10 wt % (10-100 mg g-1), and the solvent flow rate was varied between 0.1 and 0.3 mL min.-1 A minimum solvent/antisolvent flow rate ratio of 1:40 was used. Product Characterization. Particle Size and Morphology. A scanning electron microscope (Hitachi S-4500) was used to assess the particle size and morphology of the micronized PVP and the Cu-Indo/PVP coprecipitate. The samples were mounted on a metal stub and gold-coated (Polaron sputter coater) under vacuum with 40 nm of gold. Drug Loading. A known amount of coprecipitate was dissolved in methanol. The drug content in the CuIndo/PVP coprecipitate was determined by measuring the mass of indomethacin present using high-performance liquid chromatography (HPLC). The HPLC used


consisted of a Waters 600 pump, a 996 photodiode array detector, and a 717 Plus Autoinjector. The samples were analyzed using a symmetry C18 column with a mobile phase containing 70% acetonitrile and 30% 0.01 M tetramethylammonium chloride in 0.1% acetic acid. A flow rate of 1 mL min-1 was used, and the analytes were detected at a wavelength of 320 nm. The mass of indomethacin present in the sample was evaluated. The corresponding amount of Cu-Indo present in the original solution was then calculated, based on the assumption that all indomethacin present in the coprecipitate is in the form of Cu-Indo and no free indomethacin is present. Drug Stability and Residual Solvent. The stability of Cu-Indo after processing by ASES was assessed using room-temperature X-band electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC). The EPR spectroscopy was used to determine the integrity of the Cu-Indo dimer. A Bruker EMX EPR at ambient temperature and a X-band frequency of approximately 9.5 GHz was used. The spectra indicate the presence of any Cu(II) dimer, Cu(II) monomer, and paramagnetic impurities in the coprecipitate. By comparison with the X-band EPR spectra for factory-grade Cu-Indo, the stability of the drug throughout processing was examined. The DSC (2010 TA Instruments) was used for analyzing the drug stability and detecting the presence of solvent, by heating 10 mg of sample in aluminum pans from 298 K to the desired upper limit at 10 K min-1. Samples of Cu-Indo, PVP, a physical mixture of CuIndo and PVP, and a coprecipitate sample were analyzed and compared. Dissolution Study. The effect of the presence of PVP, in various ratios, on the dissolution rate of CuIndo in water was studied by powder dissolution using the USP paddle method (VK6000, Vankel Industries, Inc., Germany). The mass of the sample used in each experiment was based on having 10 mg of Cu-Indo. The dissolution medium was 1 L of deionized water (MilliQ) at 310 K, and a stirring rate of 100 rpm was used. Accurately weighed samples were added to the dissolution medium, and aliquots (approximately 4 mL) were withdrawn with a syringe from the middle of the dissolution medium at fixed time intervals. The CuIndo content in the withdrawn samples was calculated by measuring the absorbance at 240 nm (Hitachi U-2000 spectrophotometer). Solubility Study. The effect of the presence of PVP, in various ratios, on the solubility of Cu-Indo in ethanol was studied. The HPLC analytical technique, as described for drug loading, was used to determine the solubility of Cu-Indo in ethanol. An excess quantity of coprecipitate was added to 4 mL of ethanol at room temperature. The solutions were mixed using sonication. Once the solutions had equilibrated to room temperature after sonication, the excess solid was then removed by filtering the solution through a 0.45 µm syringe filter. The mass of indomethacin was then calculated using HPLC analysis, enabling the corresponding amount of Cu-Indo in the solution to be calculated and compared between samples. Results and Discussion PVP Micronization by the ASES Process. The feasibility of utilizing the ASES technique for micronization of PVP was examined. The effect of parameter

Figure 2. (a) SEM image of unprocessed PVP (10 000 MW). (b) SEM image of PVP (10 000 MW) processed by ASES (298 K, 14 MPa, 5 wt % PVP in DMF at 0.1 mL min-1, 100 µm nozzle). (c) SEM image of PVP (55 000 MW) processed by ASES (298 K, 7 MPa, 10 wt % PVP in DMF at 0.3 mL min-1, 50 µm nozzle).

variation on particle formation was studied for PVP of 10 000 MW. A comparison with a sample of 55 000 MW was also conducted. The unprocessed PVP, which is the raw material for micronization, consisted of irregular particles with a broad particle size distribution, ranging from 4 to 130 µm (Figure 2a). After ASES processing, the particle size


was dramatically decreased to less than 1 µm, as shown in Figure 2b,c. The processing of PVP with the ASES technique produced microspheres with a narrow particle size distribution and improved homogeneity. These are important features when the polymer is to be used for pharmaceutical applications. Fine particles increase the rate of dissolution because of the larger surface area, and this improves the ease of application. For consistency between doses, regular particle size and morphology are desired and are achieved by ASES processing. Trends observed because of variation of the operating parameters in the ASES process are very particular to the specific apparatus and the nature of the solvent and solute combination. The effect on the particle formation of variations in temperature, antisolvent density (pressure), solute concentration, nozzle diameter, solvent flow rate, and PVP molecular weight was found to be minimal in this system over the range studied. The precipitated particles, in general, were microspheres with a primary particle size ranging from 50 nm to 4 µm. The PVP particles produced by the ASES technique were agglomerated to some extent, though individual spheres were still evident and significant bridging between particles was not observed. At low pressure, such as 7 MPa, the particles were more agglomerated than at higher pressures, such as 14 MPa, where minimal agglomeration was observed. The lower pressure also corresponds to low antisolvent density, which means that the amount of solvent that can be extracted by carbon dioxide is decreased at low pressure. Concentration can be a dominant factor in polymer precipitations; however, variation of the PVP concentration between 1 and 10 wt % had no effect on the particle size and morphology. The lack of particle variation with concentration change indicates that the concentrations studied are below the plait point concentration of PVP. Above the plait point concentration, morphologies such as fibers can be formed by the ASES technique.18,31 Coprecipitation of PVP and Copper Indomethacin by the ASES Process. Effect of the Process Parameters on Particle Size and Morphology. The investigations into the precipitation of PVP from DMF, using the ASES process and CO2 as an antisolvent, showed that it was possible to form microspheres of PVP with a particle size of less than 1 µm when operating at subcritical conditions, such as 298 K and 14 MPa. Similarly, micronization of Cu-Indo by the ASES technique has been studied. Unprocessed Cu-Indo produced by conventional synthesis, which was the raw material for coprecipitation, had irregular particles ranging in size from 500 nm to 100 µm, as shown in Figure 3a. Cu-Indo was micronized from DMF by the ASES process at 298 K and 6.9 MPa with a solute concentration of 10 wt % (100 mg g-1). Microspheres of Cu-Indo were produced with a diameter of less than 5 µm (Figure 3b). The various morphologies formed when Cu-Indo was precipitated by the ASES process at a range of operating conditions have been previously studied.30 It is evident that ASES processing can produce a significant particle size reduction and an improvement in homogeneity in Cu-Indo particles, similar to that observed for PVP micronization. The feasibility of utilizing the ASES technique for coprecipitation of Cu-Indo with PVP was then investigated. The aims of the coprecipitation experiments were to show the applicability of ASES to produce drug/

Figure 3. (a) SEM of unprocessed Cu-Indo. (b) SEM image of Cu-Indo processed by ASES (298 K, 6.9 MPa, 10 wt % Cu-Indo in DMF at 0.2 mL min-1, 229 mm nozzle).30

polymer coprecipitates, to study the effect of PVP on Cu-Indo solubility, and to investigate the effect of process variables on the coprecipitate characteristics. Four ratios of drug to polymer were studied: 50:50, 40: 60, 30:70, and 10:90. Coprecipitation of Cu-Indo and PVP by the ASES process was conducted at an antisolvent subcritical liquid condition, 298 K and 14 MPa, a vapor-over-liquid condition, 298 K and 6.6 MPa, and a supercritical condition, 313 K and 19 MPa. At each of these conditions, microspheres were produced that ranged in size from 50 nm to 2 µm, as shown in Figure 4, with a low degree of agglomeration. The coprecipitates were pale green, suggesting that both Cu-Indo (green) and PVP (white) compounds were present. These results show that the ASES process is a feasible technique for producing microspheres of drug/polymer coprecipitates that are suitable for pharmaceutical application. Variation of the solute concentration, Cu-Indo/PVP ratio, temperature, and antisolvent density had a negligible effect on the size and morphology of the coprecipitate over the range studied. Observations of the jet during spraying at some conditions, such as when a 50 µm nozzle was used or when the solution was sprayed into the vapor region of a two-phase system, revealed that skin formation occurred around the jet. Once skin formation occurred, a


Figure 5. X-band EPR spectra for Cu-Indo and for a 50:50 CuIndo/PVP coprecipitate. Figure 4. SEM image of Cu-Indo/PVP coprecipitate formed by the ASES technique (298 K, 14 MPa, 50:50 Cu-Indo/PVP, 10 wt % solute in DMF, 0.1 mL min-1, 100 µm nozzle).

precipitate was seen to build up near the tip of the nozzle until at some critical size the large particle fell to the bottom of the vessel, producing large, irregular particles in addition to microspheres. Applying a mechanical shock to the vessel during spraying can prevent skin formation. Alternatively, a vibrating nozzle could be used because it aids in jet breakup and may prevent fiber formation. The formation of fibers by ASES is commonly observed when spraying polymer solutions of high concentration.18,31 At high polymer concentrations, jet breakup is prevented because of increased solution viscosity. At higher solute concentrations, less antisolvent is required to diffuse into the solution to initiate nucleation and precipitation occurs earlier, further preventing jet breakup. As a result, skin formation may occur around the nozzle and continuous polymer morphologies such as fibers are formed. Determination of Drug Loading of the Coprecipitate. The drug loading of the coprecipitate produced by the ASES process, at different Cu-Indo to PVP ratios and two different pressures, was measured. The coprecipitate drug content was then compared to the composition of the sample prior to processing. The concentration of Cu-Indo in the coprecipitate was higher than expected, by an average of 6%, in all cases except one, where the drug content was equal to the expected value. The reason for the slightly higher Cu-Indo content may be due to loss of polymer if some of the PVP is extracted by carbon dioxide during the precipitation. PVP has a high solubility in DMF, and a small amount of DMF in the vessel may be enough to dissolve some of the polymer. The higher proportion of Cu-Indo in the product may also be due to solvent removal. The drug contains residual solvent from synthesis and two solvent ligands in its structure. The ASES process acts as a purification technique, and therefore the coprecipitate may have a higher proportion of Cu-Indo because the drug weighs less after ASES processing as a result of loss of solvent. Effect of ASES Processing on Drug Stability. A major concern in the processing of Cu-Indo is that the drug may dissociate and produce indomethacin, which is toxic. The drug loading study indicates that the same relative amounts of indomethacin are present as there were in the sample before processing.

The presence of copper was detected using X-band EPR to investigate the nature of the coprecipitate. The X-band EPR spectra, at room temperature, for the factory-grade Cu-Indo and the coprecipitate with a 50: 50 drug-to-polymer ratio is shown in Figure 5. Two peaks dominate the spectra of the coprecipitate. The peak at approximately 4700 G indicates the presence of a Cu(II) dimer [Cu2(Indo)4(DMF)2]. The prominent peak on the X-band EPR spectrum for the factorygrade Cu-Indo represents the same Cu(II) dimer. The dimer feature is also observed at 500 and 5980 G in both samples. The peak observed in the Cu-Indo and coprecipitate spectra at approximately 3300 G indicates the presence of a Cu(II) monomer. The Cu(II) monomer peak is significantly larger in the coprecipitate compared to the factory-grade Cu-Indo. The amount of monomer is unquantified; however, it is evident that although a significant amount of dimer is present, some of the drug is converted to monomer. The fact that a larger percentage of the Cu(II) monomer is present in the Cu-Indo/PVP coprecipitate is of concern because the active form of Cu-Indo is thought to be the dimeric complex.32 The stability trial suggests that the indomethacin quantified in the drug loading study is present as both Cu(II) dimer and Cu(II) monomer because no other copper compounds were detected. Drug stability and the presence of residual solvent were investigated by analyzing the physical properties of the drug, the polymer, a physical mixture of drug and polymer in a 50:50 ratio, and the 50:50 coprecipitate using DSC analysis. The DSC spectra obtained for each of these samples are shown in Figure 6. Cu-Indo has two prominent endothermic changes. The broad peak between 373 and 423 K corresponds to loss of solvent because DMF has a boiling point of 426 K. The presence of DMF is expected because it is used in the synthesis of Cu-Indo and is present in the Cu-Indo molecular structure. The peak at 478 K corresponds to the melting point of Cu-Indo. The melting peak is well-defined for Cu-Indo because it is crystalline, and therefore more energy is needed in melting. The remaining peaks are decomposition peaks. The DSC spectrum of PVP does not show a melting point at 573 K, which is the expected melting point of PVP (10 000). The absence of a melting point peak is most likely due to the amorphous nature of the polymer and the correspondingly low energy required for a phase transition. The broad peak from ambient to 373 K can be attributed to loss of water. The DSC spectrum of


Figure 7. Dissolution profile of Cu-Indo in water: (b) Cu-Indo; (O) micronized Cu-Indo; (×) 50:50 physical mixture; ([) 10:90 Cu-Indo/PVP coprecipitate; (2) 50:50 Cu-Indo/PVP coprecipitate; (9) 30:70 Cu-Indo/PVP coprecipitate.

Figure 6. DSC thermogram for PVP, Cu-Indo, a 50:50 Cu-Indo/ PVP physical mixture, and a 50:50 Cu-Indo/PVP coprecipitate.

PVP is in agreement with that which had been previously reported by other authors.8,33 The DSC spectrum for the physical mixture of CuIndo and PVP included the same peaks as those of each of the individual components. These results indicate that the crystal structure of the polymer and drug has not changed and that they are still separate entities. As can be seen in Figure 6, the DSC spectrum for the coprecipitate is significantly different from that of the physical mixture. The solvent loss peak is not present in the coprecipitate, suggesting that ASES processing has purified the drug and may have eliminated the DMF from the molecular structure. It was also observed that the melting point of Cu-Indo is lower in the coprecipitate. If the PVP has formed a complex with the CuIndo, then this new complex may have a melting point slightly lower than the original drug. The results from the DSC analysis indicate that the Cu-Indo/PVP coprecipitate is not simply a mixture of the two compounds. Effect of Coprecipitation on Drug Dissolution Rate. The dissolution rates in water of unprocessed Cu-Indo, micronized Cu-Indo, a physical mixture of PVP and Cu-Indo, and Cu-Indo/PVP coprecipitates produced by ASES are compared in Figure 7. The dissolution rate coefficient, Kw, was used to compare dissolution rates and is equal to the inverse of the time taken for 63.2% of the sample to dissolve. The dissolution rate coefficients of the micronized Cu-Indo and the 10:90, 30:70, and 50:50 Cu-Indo/PVP coprecipitates were calculated to be 0.015, 0.015, 0.038, and 0.016 s-1, respectively. The value of the dissolution rate coefficient could not be calculated for the physical mixtures or the factory-grade Cu-Indo because those samples did not attain 63.2% dissolution in the time period used for the study, indicating that the rate coefficient for these samples is less than 0.011 s-1. It is evident from Figure 7 and the Kw values that the dissolution rates of micronized Cu-Indo and the Cu-Indo/PVP coprecipitates were dramatically enhanced compared to those of the factory-grade Cu-Indo or the Cu-Indo/PVP physical mixture. The dissolution

profiles of micronized Cu-Indo and Cu-Indo/PVP coprecipitates were similar. No significant difference in the dissolution rate was observed between Cu-Indo and a 50:50 physical mixture of Cu-Indo and PVP. The increase in the dissolution rate for the micronized Cu-Indo and coprecipitate samples compared to that of the factory-grade Cu-Indo is due to the decreased particle size achieved by ASES processing. The factorygrade Cu-Indo ranged in particle size from 500 nm to 100 µm, and ASES processing produced particles ranging from 50 nm to 5 µm. The coprecipitate with a drug-to-polymer ratio of 30: 70 averaged a substantially higher dissolution rate coefficient than the 50:50 or 10:90 samples. Therefore, as an increasing amount of PVP is added to Cu-Indo, the dissolution rate increases to a maximum and then decreases. The maximum in the dissolution rate has been previously observed for PVP coprecipitated with frusemide,11 potassium chloride,34 and the anti-inflammatory CI-987.15 Kearney et al. observed a maximum dissolution rate, 15 times faster than that of the pure CI-987, with a PVP weight fraction of 0.81.15 Kearney et al. suggest that this trend is due to the decrease in crystallinity at higher PVP fractions and the maximum occurs when the drug exists in a totally amorphous form. As the weight fraction of PVP is increased further than the point where the coprecipitate is completely amorphous, the dissolution of PVP becomes the rate-controlling step and the dissolution rate is decreased.15 Grassi et al. state that the solubility of amorphous drugs in hydrophilic fluids, such as water and physiological fluid, is usually higher than that of crystalline drugs.35 Therefore, the addition of polymer to drugs can significantly increase the dissolution rate because an amorphous drug state is eventually attained. No significant increase in the solubility was observed when the Cu-Indo/PVP coprecipitates were dissolved in water; therefore, it is unlikely that the increase in the dissolution rate was a result of an increased solubility of Cu-Indo in water. After 1 h, approximately 8%, 14%, 60%, and 65% of the drug in each of the Cu-Indo, 50:50 Cu-Indo/PVP physical mixture, micronized Cu-Indo, and coprecipitate (average for all Cu-Indo/PVP ratios) had dissolved, respectively. It can therefore be seen that the rate of


dissolution of the PVP/Cu-Indo coprecipitate is at least 8 times faster than that of factory-grade Cu-Indo. These results show that micronization will improve the dissolution rate of Cu-Indo significantly and it may be further improved by the presence of PVP. Effect of Coprecipitation on the Solubility of Cu-Indo. The study was conducted to investigate the potential of using PVP to increase the solubility of CuIndo in biologically nontoxic solvents. The solubilities of factory-grade Cu-Indo, a physical mixture of CuIndo and PVP, and each coprecipitate ratio were measured and compared in ethanol. A solubility enhancement factor was defined as the ratio of the mass of CuIndo that could be dissolved in a given volume of ethanol from each sample to the amount of unprocessed CuIndo that could be dissolved in the same volume of ethanol. The solubility enhancement factor was calculated to give the effect of PVP on Cu-Indo solubility a relative value. A significant enhancement in the solubility of CuIndo in ethanol was observed for the Cu-Indo/PVP coprecipitates obtained from the ASES process. The greatest enhancement, a factor of 93.7, was observed for the Cu-Indo/PVP coprecipitate with a drug-topolymer ratio of 10:90. As the ratio of Cu-Indo to PVP increased, the solubility enhancement factor decreased to 5.3 and 3.3 for the 30:70 and 50:50 coprecipitates, respectively. No significant solubility enhancement was observed for the Cu-Indo/PVP physical mixtures. The actual interaction between the polymer and drug, which enables the solubility enhancement, has not been confirmed. Two possible hypotheses have been presented to explain the solubility enhancement observed when PVP is coprecipitated with compounds of lower molecular weight. The first hypothesis explains the solubility enhancement in terms of an interaction between the hydrocarbon segments of the polymer and similar portions of the cosolute.36,37 The interaction, termed the “hydrophobic bond” concept, has been used to explain the increase in solubility observed for PVP/ cosolute coprecipitates.9 The second hypothesis explains the solubility enhancement in terms of the formation of a complex between the drug and PVP.8,13 The formation of a complex has been used to explain the solubility enhancement observed for a PVP/sulfathiazole coprecipitate.13 The complex was proposed to have formed by hydrogen bonding between the amino groups of the sulfathiazole and the oxygen groups of the PVP. The results from the DSC analysis indicated that the Cu-Indo/PVP coprecipitate is not simply a mixture of the two compounds. The results support the hypothesis that the increase in Cu-Indo solubility in ethanol is due to complex formation between PVP and Cu-Indo during coprecipitation. The loss of the solvent peak in the DSC spectrum of the coprecipitate, shown in Figure 6, indicates that the coordination site on the copper that is normally occupied by a solvent molecule may be occupied by PVP. The presence of a larger Cu(II) monomer signal in the X-band EPR spectrum (Figure 5) suggests that PVP may also be interfering with the Cu-Cu bond of the Cu-Indo. A pharmaceutical drug is suitable for parenteral applications if it has a relatively high solubility in a suitable solvent. The solvent most commonly used for administration by injection is water. While the solubility of Cu-Indo in water has not been significantly enhanced by coprecipitation, the enhancement in solubility

in ethanol is dramatic. Therefore, an ethanol/water combination may be able to be used to solubilize CuIndo to an extent suitable for parenteral administration, while remaining within the levels of safe solvent administration. The amount of ethanol required to administer the drug must be minimized because large amounts may be harmful. The required dosage of Cu-Indo is 0.2 mg kg-1 of body weight. When it is supposed that the average mass of a horse is 450 kg, 90 mg of Cu-Indo is required per dose. Therefore, to administer the required dose, at least 58 mL of an ethanol solution of unprocessed Cu-Indo would be needed. By formation of a Cu-Indo/PVP coprecipitate, the increase in solubility of the drug reduces the amount of ethanol required to 17, 11, and 0.6 mL for the 50:50, 70:30, and 90:10 coprecipitates, respectively. These values are the minimum amounts of ethanol that would need to be administered because they are based on a saturated solution. The saturated solution of 10:90 Cu-Indo/PVP coprecipitate in ethanol was extremely viscous because of the high PVP content. Because the desired application is for injection, a solution with high viscosity is not practical because it is difficult to inject with a syringe. Therefore, it is necessary to find the optimal condition between increased solubility of Cu-Indo, minimization of the ethanol volume, and viscosity of the solution. There are also limitations to the amount of PVP that can be delivered to the body that must be considered when determining the optimum ratio. For intramuscular use, PVP should be injected only into muscle masses with sufficient blood supply, to a maximum concentration of 100 mg/injection and limited to alternative sites for up to 30 days.7 For intravenous use, it can be continuously administered for periods of up to 90 days at dosage levels not to exceed 100 mg dose-1.7 Subcutaneous administration is not recommended. Therefore, the optimum coprecipitate examined is that with a Cu-Indo to PVP ratio of 50:50 because the coprecipitates with higher polymer content would exceed the limit of 100 mg of PVP per dose. Use of a Cu-Indo/ PVP coprecipitate with a drug-to-polymer ratio of 50: 50 improves the solubility while maintaining a workable viscosity. The improvement in the Cu-Indo solubility and subsequent reduction of the amount of ethanol required per dose may make it possible to deliver CuIndo by the parenteral route in a single administration. The stability of Cu-Indo/PVP coprecipitates in ethanol was studied at room temperature and showed that the solution is not stable for a long time period. Depending on the concentration of the sample in solution, after a given time period, precipitation will occur. The precipitation may be due to the ethanol replacing the PVP on the Cu-Indo ligand site, causing the CuIndo to revert to the slightly soluble form and the excess solute to precipitate. At concentrations of 1.5 and 4.6 mg of Cu-Indo/mL of ethanol, the coprecipitate with a drug-to-polymer ratio of 10:90 will remain in solution for 78 and 3 h, respectively, before precipitation begins at ambient conditions. Therefore, the high-concentration solution must be injected to the body within 3 h of being made. As the concentration of Cu-Indo in the sample was increased, the time the solution remained stable decreased. Depending on the concentration, it is therefore recommended that the solution is made prior to injection and not left for a long period. Once the drug


enters the body, it is changed immediately because it is diluted by the blood and metabolized. Conclusions The study has shown that it is possible to coprecipitate Cu-Indo and PVP by the ASES technique and produce homogeneous microparticles suitable for pharmaceutical application. It is evident that both micronization and coprecipitation of Cu-Indo increase its rate of dissolution in water. The benefit of ASES processing is that it can micronize and coprecipitate in one step. The advantage of coprecipitation over micronization is that coprecipitation of Cu-Indo with PVP not only increases the rate of dissolution but also increases the solubility. Acknowledgment The authors gratefully acknowledge the financial support of the CRC for Polymers. The support provided by the Australian Research Council, under the auspice of the SPIRT Program (C89917624), and the Australian Government, for provision of an Australian Postgraduate Research Award (B.W.) and an Australian Research Council Postdoctoral Fellowship (F.D.), is gratefully acknowledged. Literature Cited (1) Weder, J. E.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; MacLachlan, D.; Bramley, R.; Delfs, C. D.; Murray, K. S.; Moubaraki, B.; Warwick, B.; Biffin, J. R.; Regtop, H. L. Antiinflammatory Dinuclear Copper(II) Complexes with Indomethacin. Synthesis, Magnetism and EPR Spectroscopy. Crystal Structure of the N,N-Dimethylformamide Adduct. Inorg. Chem. 1999, 38 (8), 1736. (2) Weder, J. E.; Hambley, T. W.; Kennedy, B. J.; Lay, P. A.; Foran, G. J.; Rich, A. M. Determination of the Structures of Antiinflammatory Copper(II) Dimers of Indomethacin by MultipleScattering Analyses of X-ray Absorption Fine Structure Data. Inorg. Chem. 2001, 40 (6), 1295. (3) Warwick, B.; Dehghani, F.; Foster, N. R.; Biffin, J. R.; Regtop, H. L. Synthesis, Purification, and Micronization of Pharmaceuticals Using the Gas Antisolvent Technique. Ind. Eng. Chem. Res. 2000, 39 (12), 4571. (4) Engwicht, A.; Girreser, U.; Muller, B. W. Critical Properties of lactide-co-glycolide Polymers for the use in Microparticle Preparation by the Aerosol Solvent Extraction System. Int. J. Pharm. 1999, 185 (1), 61. (5) Young, T. J.; Johnston, K. P.; Mishima, K.; Tanaka, H. Encapsulation of Lysozyme in a Biodegradable Polymer by Precipitation with a Vapor-over-Liquid Antisolvent. J. Pharm. Sci. 1999, 88 (6), 640. (6) Ghaderi, R.; Artursson, P.; Carlfors, J. A New Method for Preparing Biodegradable Microparticles and Entrapment of Hydrocortisone in DL-PLG Microparticles Using Supercritical Fluids. Eur. J. Pharm. Sci. 2000, 10, 1. (7) Tableting with Plasdone. International Specialty Products Corp., 1994, Povidone USP. (8) Rodriguez-Espinosa, C.; Martinez-Oharriz, M. C.; Martin, C.; Goni, M. M.; Velaz, I.; Sanchez, M. Dissolution Kinetics for Coprecipitates of Diflunisal with PVP K30. Eur. J. Drug Metab. Pharmacokinet. 1998, 23 (2), 109. (9) Svoboda, G. H.; Sweeney, M. J.; Walkling, W. D. Antitumor Activity of an Acronycine-Poly(vinylpyrrolidone) Coprecipitate. J. Pharm. Sci. 1971, 60 (2), 333. (10) Tantishaiyakul, V.; Kaewnopparat, N.; Ingkatawornwong, S. Properties of Solid Dispersions of Piroxicam in Polyvinylpyrrolidone. Int. J. Pharm. 1999, 181 (2), 143. (11) Doherty, C.; York, P. Mechanisms of Dissolution of Frusemide/PVP Solid Dispersions. Int. J. Pharm. 1987, 34 (3), 197. (12) Stupak, E. I.; Bates, T. R. Enhanced Absorption and Dissolution of Reserpine from Reserpine-Poly(vinylpyrrolidinone) Coprecipitates. J. Pharm. Sci. 1972, 61 (3), 400.

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Received for review June 9, 2003 Revised manuscript received October 17, 2003 Accepted November 7, 2003 IE030483Q

Increasing Copper Indomethacin Solubility by Coprecipitation with

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