Investigation of drying stresses on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer

This article describes the design, performance testing, and application of a controlled humidity mini-freeze-dryer in studying the physical stability of lactate dehydrogenase during lyophilization. Performance evaluation of the mini-freeze-dryer was conducted with tests, namely water sublimation, radiation heat exchange, lowest achievable temperature, and leak testing. Protein stability studies were conducted by comparing protein activity at various stages of lyophilization with the initial activity. The shelf and condenser temperature were stable at <-40 degrees C, wall temperature was within 2 degrees C of the shelf temperature, and the leak rate was small. The chamber pressure was controlled by the ice on the condenser and the product temperature during sublimation was equal to the shelf temperature. Addition of Tween 80 prevented activity loss in solution and after freeze-thaw. No activity loss was observed after primary-drying even in absence of lyoprotectants and with collapse of cake structure. Five percent (w/w) sucrose concentration was required to achieve full stabilization. In conclusion, performance testing established that the mini-freeze-dryer was suitable for mechanistic freeze-drying studies. Secondary-drying was the critical step for protein stability. The concentration of sucrose required to stabilize the protein completely was several orders of magnitude higher than that required to satisfy the direct interaction requirement of the protein.     

Cake shrinkage during freeze drying: a combined experimental and theoretical study

The aim of this study is to investigate, by experimental studies and theoretical analysis, the phenomenon of cake shrinkage during the lyophilization process and to investigate the effect of shelf temperature during primary drying and secondary drying on the degree of cake shrinkage. Freeze-drying experiments were performed using 5% w/v sucrose where the drying protocols were altered in order to produce differing product temperature profiles. Resistance data during freeze drying were evaluated by the Manometric Temperature Measurement (MTM) method. Theoretical simulation of the freeze-drying process was performed using the Passage Freeze-Drying software. The difference between the glass transition temperature and the product temperature (Tg-T) obtained from the theoretical analysis was calculated and used for correlation with experimental shrinkage data. The Brunauer, Emmeth, Teller (BET) Specific Surface Area (SSA) Analysis was used as a method to quantify the degree of shrinkage. Samples were also analyzed for pore volume by mercury porosimetry. The SSA analysis on the freeze-dried samples showed an increase in SSA when samples were freeze dried at a lower shelf temperature during primary drying and at a slower ramp rate during secondary drying. The trend in surface area values was consistent with that obtained for pore size values. However, differences obtained among the various samples are small and cake diameter measurements showed that there was approximately 17% shrinkage even in the sample freeze dried at temperatures well below the Tg' and Tg. Variations in process and product temperature only accounted for an additional 2%-3% shrinkage. Resistance data obtained at various primary drying shelf temperatures showed a good correlation with surface area. The Tg-T behavior of the freeze-dried samples showed that a slow ramp rate of 0.1 degrees C/min during secondary drying maintains a product well below the Tg at all times and a higher ramp rate gives negative values of Tg-T. The obtained data suggest that conditions of secondary drying do impact shrinkage, and it is important to maintain a sample well below the collapse temperature during primary drying and below the Tg at all times during secondary drying; however, drying conditions are a second order effect. It seems that, in the case of a sample like sucrose, nearly 17% shrinkage will occur no matter what the product temperature history.     

Stabilization of IgG by supercritical fluid drying: optimization of formulation and process parameters.

The aim of this study was to stabilize human serum immunoglobulin G (IgG) by a supercritical fluid (SCF) drying process. Solutions containing IgG (20mg/ml) and trehalose or hydroxypropyl-beta-cyclodextrin in a 1:4 (w/w) ratio were sprayed into a SCF phase consisting of CO(2) and ethanol at 100bar and 37 degrees C. Initially, a set of drying conditions previously developed to successfully stabilize lysozyme and myogobin formulations was used [N. Jovanovi?, A. Bouchard, G.W. Hofland, G.J. Witkamp, D.J.A. Crommelin, W. Jiskoot, Eur. J. Pharm. Sci. 27 (2006) 336-345]. Dried formulations were analyzed by Karl Fisher titration, scanning electron microscopy, X-ray powder diffraction, and modulated DSC. Protein structure in the solid-state was studied by FTIR and after reconstitution by UV/Vis, circular dichroism and fluorescence spectroscopy, GPC and SDS-PAGE. When IgG was dried under the above-mentioned conditions, substantial amounts of insoluble aggregates were formed. Addition of buffer helped to reduce the fraction of insoluble material but not of soluble aggregates. Full stabilization could be achieved by adjusting the process conditions: drying without ethanol while keeping the other conditions the same, or drying with ethanol at a temperature below the critical point (20 degrees C). In conclusion, it is possible to stabilize human IgG by SCF drying provided that the formulation and process conditions are tailored to meet the specific requirements of the protein.     

A pilot study of freeze drying of poly(epsilon-caprolactone) nanocapsules stabilized by poly(vinyl alcohol): formulation and process optimization

A common limitation of using polymeric nanoparticles in aqueous suspension is due to their poor chemical and physical stability when conserved for a long time. Therefore, freeze drying of these colloidal systems is an alternative method to achieve long-term stability. Nanocapsules have thin and fragile shell structure, which may not resist to the stress of such process. The aim of this study is to investigate the formulation and process parameters in order to ensure the stability of polycaprolactone nanocapsules (PCL NC) by freeze drying. In this paper, we studied the freeze drying of PCL NC prepared by the emulsion-diffusion method and stabilized by poly(vinyl alcohol) (PVA). Different parameters have been tested throughout the freeze-thawing study including PVA and PCL concentration, cooling rate, cryoprotectant concentrations, nature of encapsulated oil and NC purification. On the other hand, nanocapsules have been freeze dried both before and after purification. Freeze dried purified PCL NC were characterized by particle size measurement, collapse temperature, T'g determination, scanning electron microscope observation, environmental scanning electron microscope imaging and residual humidity quantification. Finally, the effect of annealing on the NC stability and the sublimation rate has been well explored. The results suggest that PCL NC could be freeze dried without a cryoprotectant if the concentration of PVA stabilizer is sufficient (5%), while for the purified NC the addition of 5% of cryoprotectant seems to be necessary to ensure the stability of NC. The type of cryoprotectants had practically negligible effects on the size and the rehydration of freeze dried nanocapsules. The annealing process could accelerate the sublimation with the conservation of nanocapsules size.     

Freeze drying of nanosuspensions, 2: the role of the critical formulation temperature on stability of drug nanosuspensions and its practical implication on process design

The present study investigates whether controlling the product temperature below the critical formulation temperature (CFT) during primary drying in a freeze drying cycle is a prerequisite for the stabilization of drug nanoparticles. For that purpose, the CFT of four drug nanosuspensions stabilized with different types (amorphous and crystalline) and concentrations of steric stabilizers and either of the disaccharides, trehalose and sucrose, was determined by differential scanning calorimetry and freeze‐dry microscopy. Freeze‐drying experiments were performed such that product temperatures during primary drying remained either below or well above the CFT of individual mixtures. It was found that glass formation did not influence the stability of the nanoparticles, suggesting that an adequate type of steric stabilizer and lyoprotectant concentration is present. Freeze drying could also be performed above the eutectic temperature without compromising on the final product quality profile, such as nanoparticle size and structural preservation of the lyophilized cake. The high concentration of solid drug nanoparticles provided additional cake stability. The results of this study confirm for the first time that primary drying for drug nanosuspensions can be greatly shortened because induced viscous flow or even meltback is not a limitation for nanoparticle stability and cake elegancy. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4471–4481, 2011     

A comparative study on the physicochemical and biological stability of IgG1 and monoclonal antibodies during spray drying process

Background

The main concern in formulation of antibodies is the intrinsic instability of these labile compounds. To evaluate the physicochemical stability of antibody in dry powder formulations, physical stability of IgG₁ and a monoclonal antibody (trastuzumab) during the spray drying process was studied in a parallel study and the efficacy of some sugar based excipients in protection of antibodies was studied.

Results

The SDS-PAGE analysis showed no fragmentation of antibodies after spray drying in all formulations. The secondary structure of antibodies contained 40.13 to 70.19% of β structure in dry state. Also, CD spectroscopy showed the similar secondary structure for trastuzumab after reconstitution in water. ELISA analysis and cell culture studies were conducted in order to evaluate bioactivity of monoclonal antibody. Formulations containing combination of excipients provided maximum tendency of trastuzumab to attach to the ELISA antigen (86.46% ± 2.3) and maximum bioactivity when incubated with SKBr₃ cell line (the cell viability was decreased to 65.99% ± 4.6). Incubation of formulations with L929 cell line proved the biocompatibility of the excipients and non-toxic composition of formulations.

Conclusion

The IgG₁ and trastuzumab demonstrated similar behavior in spray drying process. The combination of excipients containing trahalose, hydroxypropyl beta cyclodextrin and beta cyclodextrin with proper ratio improved the physical and chemical stability of both IgG₁ and monoclonal antibody.     

Study of the individual contributions of ice formation and freeze-concentration on isothermal stability of lactate dehydrogenase during freezing

The objective of this study was to determine the individual contributions of ice formation, solute concentration, temperature, and time, to irreversible protein denaturation during freezing. A temperature-step approach was used to study isothermal degradation of frozen lactate dehydrogenase (LDH). The freeze-concentrate composition was determined using differential scanning calorimetry to enable preparation of solutions, without ice, of the same concentration as the freeze-concentrate, and thereby determine the role of the freeze-concentrate composition on LDH degradation. Both stabilizers employed in the study, hydroxyethyl starch and sucrose, conferred cryoprotection on LDH. While LDH stability was lower at 1.50-3.25% w/v sucrose than in the absence of sucrose, cryoprotection was restored at higher sucrose concentrations. pH shift during freezing, degree of supercooling, and excipient impurities were ruled out as causes for unusual LDH stability behavior at lower sucrose concentrations. Specific surface area measurements of the freeze-dried cakes showed that the ice surface area increased with an increase in sucrose concentration. No LDH degradation occurred in concentrated solutions, without ice, at the same composition as the freeze-concentrate in frozen systems where massive degradation was documented. Thus, ice formation is the critical destabilizing factor during freezing of LDH in sucrose:citrate buffer systems.     

Through-vial impedance spectroscopy of critical events during the freezing stage of the lyophilization cycle: The example of the impact of sucrose on the crystallization of mannitol

The aim of this work was to evaluate the application of through-vial impedance spectroscopy in the measurement of eutectic crystallization during the freezing stage of the lyophilisation cycle.Impedance measurements of various sugar solutions (mannitol 5%, 10% and 15% w/v, sucrose 5% w/v and mannitol 5% w/v, and sucrose 5% w/v solutions) were taken during a freeze–thaw cycle, over a frequency range 10–10⁶ Hz with a scan interval of 1.5 min, using measurement vials with externally attached electrodes connected to a high resolution impedance analyzer.Estimates for the electrical resistance of the mannitol solutions record the exothermic crystallization of mannitol at a temperature of −24 °C during the temperature ramp down stage of the freezing cycle, which is in close agreement with the off-line DSC measurement of −22 °C. The freezing profile of a 5% mannitol solution with 5% sucrose (a component that does not crystallize in the frozen solution) demonstrated the inhibition of mannitol crystallization (with the implication that the product will then require sub-T_g′ freezing and drying).The work suggests a role for through-vial impedance spectroscopy in the concurrent development of the product formulation and freeze drying cycle without the uncertainty introduced when using off-line date to define the critical process parameters.     

Experimental study of the impact of annealing on ice structure and mass transfer parameters during freeze-drying of a pharmaceutical formulation

The principal aim of this study is to evaluate the influence of annealing on structural properties and mass transfer parameters of a model formulation used for freeze-drying of pharmaceutical proteins. Characterization runs on ice morphology of the frozen material were performed in a cold chamber by direct optical microscopy using episcopic coaxial lighting. Consequently, it was possible to quantify the influence of annealing (temperature, duration) on ice morphology. Then, the pressure rise analysis method was used during primary drying to identify as a function of time both the dried layer mass transfer resistance values, R(p), and the sublimation front temperature, T(i), with or without annealing. Annealing turned out to accelerate sublimation rates by increasing ice crystal sizes of the frozen matrix, thus leading to lower values of R(p). Finally, secondary drying kinetics were studied by measuring the values of the desorption constant by a simple gravimetric method. In this study, annealing turned out to decrease desorption kinetics by a factor of two.     

Controlled release of a self-emulsifying formulation from a tablet dosage form: stability assessment and optimization of some processing parameters

The objective of this study was to evaluate the effect of some processing parameters on the release of lipid formulation from a tablet dosage form. A 17-run, face-centered cubic design was employed to evaluate the effect of colloidal silicates (X(1)), magnesium stearate mixing time (X(2)), and compression force (X(3)) on flow, hardness, and dissolution of Coenzyme Q(10) (CoQ(10)) lipid formulation from a tablet dosage form. The optimized formulation was subsequently subjected to a short-term accelerated stability study. All preparations had a flowability index values ranging from 77 to 90. The cumulative percent of CoQ(10) released within 8h (Y(5)) ranged from 40.6% to 90% and was expressed by the following polynomial equation: Y(5)=49.78-16.36X(1)+2.90X(2)-3.11X(3)-0.37X(1)X(2)+1.06X(1)X(3)-1.02X(2)X(3)+11.98X(1)(2)+10.63X(2)(2)-7.10X(3)(2). When stored at 4 degrees C, dissolution rates were retained for up to 3 months. Storage at higher temperatures, however, accelerated lipid release and caused leakage, and loss of hardness. Processing parameters have a profound effect on the release of lipid formulations from their solid carriers. While optimized controlled release formulations could be attained, further considerations should be made to prepare "liquisolids" that are physically stable at higher storage temperatures.     

Slow motion picture of protein inactivation during single-droplet drying: a study of inactivation kinetics of L-glutamate dehydrogenase dried in an acoustic levitator

A novel technique is presented to allow measurement of the kinetics of protein inactivation during drying of an acoustically levitated single droplet. Droplets/particles are removed from the acoustic field after various times during drying, and the state of the protein within them is analyzed. The influence of drying air temperature, relative humidity, buffer concentration, and the presence of a substrate on the inactivation of glutamate dehydrogenase is described. The kinetics of inactivation showed three distinct phases. The first phase of constant drying rate demonstrated little protein inactivation in the solution droplet. After the critical point of drying, a second phase was distinguishable when the surface temperature has risen sharply, but there is still only little inactivation of the protein in the solid particle. An onset point of rapid inactivation of the protein marked the start of the third phase that proceeded with approximately first-order rate kinetics. In the case of L-glutamate dehydrogenase, the evidence suggests that the residual moisture content of the solid and not the temperature alone determines the point of onset of protein inactivation.     

In situ precipitation and vacuum drying of interferon alpha-2a: development of a single-step process for obtaining dry, stable protein formulation

Feasibility studies were performed to develop a process for obtaining stable dry protein formulations based on in situ polyethylene glycol (PEG)-induced precipitation and vacuum drying of interferon alpha-2a (IFNalpha2a) solution in a vial. Using a laboratory scale freeze dryer, the process was carried out in two phases: first, protein solution containing PEG was concentrated to achieve protein precipitation, and second, remaining water was removed by further reducing the chamber pressure. Drying conditions, i.e. temperature and pressure, and solution composition were selected to ensure maximal precipitation (solubility of IFNalpha2a), to achieve precipitation without boiling, and to ensure stability. Dried formulations were subjected to stability studies (40 degrees C). Concentration and precipitation could be achieved at a fast rate by utilizing pressures slightly above the vapor pressure of water. Fluorescence and circular dichroism (CD) studies showed that precipitated IFNalpha2a maintained its native structure. Fourier transform infrared spectroscopy (FTIR) studies showed that IFNalpha2a when dried in the presence of trehalose, maintained its secondary structure. Trehalose also prevented formation of aggregates during drying. Moisture contents of 1% (w/w) were achieved within 48 h of drying. Dry formulation containing 1:20:100 (w/w) IFNalpha2a:trehalose:mannitol was stable against aggregation and oxidation (6% oxidized at 40 degrees C, 6 months). Stability profile was comparable to a similar lyophilized formulation.     

Investigation of physicochemical factors affecting the stability of a pH-modulated solid dispersion and a tablet during storage

The stability of solid dispersions (SD) during storage is of concern. We prepared the pH-modulated SD (pSD) and compressed tablets consisting of polyethylene glycol (PEG) 6000 as a carrier, drug and MgO (alkalizer). Telmisartan (TEL), an ionizable poorly water-soluble drug, was chosen as a model drug. The changes in physicochemical factors such as the dissolution rate, drug crystallinity, microenvironmental pH (pH(M)) and intermolecular interactions of the pSD and the tablets were investigated over 3 months under different temperature and relative humidity (RH) conditions: refrigerator (5-8 °C), 25 °C/32% RH, 25 °C/55% RH, 25 °C/75% RH, 40°C/32% RH, 40 °C/55% RH, and 40 °C/75% RH. Differential scanning calorimetry (DSC) analysis of all samples revealed no distinct changes in the drug melting point. In contrast, powder X-ray diffraction (PXRD) diffractograms revealed that samples stored at 40 °C/75% RH for 1 month, 25 °C/75% RH for 3 months and 40 °C at all humidity conditions for 3 months showed gradual recrystallization of the drug. Fourier transform infrared (FTIR) spectra indicated a reduced intensity of intermolecular interactions between TEL and MgO in the pSD and tablet. The pH(M) also gradually decreased. These altered physicochemical factors under the stressed conditions resulted in decreased dissolution profiles in intestinal fluid (pH 6.8). In contrast, the dissolution rate in gastric fluid (pH 1.2) was almost unchanged because of the high intrinsic solubility of TEL at this pH.     

Inhibitory effect of ebselen on lactate dehydrogenase activity from mammals: a comparative study with diphenyl diselenide and diphenyl ditelluride

Ebselen is a seleno compound whose antioxidant properties have been attributed to its thiol-peroxidase and thioredoxin-like activity. However, the excessive oxidation of thiols can be potentially toxic. Thus, this work investigated whether lactate dehydrogenase (LDH) can be a possible in vitro target to the toxicity of ebselen, in comparison with diphenyl diselenide [(PhSe)(2)] and diphenyl ditelluride [(PhTe)(2)]. Ebselen was the most potent inhibitor of LDH. A maximal inhibitory effect was obtained at 2 μM to LDH purified and at 20 μM to LDH from heart and liver homogenates. Moreover, (PhSe)(2), followed by (PhTe)(2), also presented a significant inhibitory effect on LDH activity. DL-dithiothreitol (DTT) was able to revert the inhibition of LDH induced by all compounds tested, confirming the involvement of essential thiol groups on LDH inhibition by organochalcogens. In conclusion, our results show that liver and heart LDH may be a possible target for the toxicity of organochalcogens at relative low concentrations. Our results also indicate that the use of LDH, as a marker of cell viability, may be biased by a direct inhibitory effect of ebselen or other chalcogenides on LDH, resulting in false protection in an in vitro system.     

Inhibitory effect of ebselen on lactate dehydrogenase activity from mammals: a comparative study with diphenyl diselenide and diphenyl ditelluride

Ebselen is a seleno compound whose antioxidant properties have been attributed to its thiol-peroxidase and thioredoxin-like activity. However, the excessive oxidation of thiols can be potentially toxic. Thus, this work investigated whether lactate dehydrogenase (LDH) can be a possible in vitro target to the toxicity of ebselen, in comparison with diphenyl diselenide [(PhSe)₂] and diphenyl ditelluride [(PhTe)₂]. Ebselen was the most potent inhibitor of LDH. A maximal inhibitory effect was obtained at 2 μM to LDH purified and at 20 μM to LDH from heart and liver homogenates. Moreover, (PhSe)₂, followed by (PhTe)₂, also presented a significant inhibitory effect on LDH activity. DL-dithiothreitol (DTT) was able to revert the inhibition of LDH induced by all compounds tested, confirming the involvement of essential thiol groups on LDH inhibition by organochalcogens. In conclusion, our results show that liver and heart LDH may be a possible target for the toxicity of organochalcogens at relative low concentrations. Our results also indicate that the use of LDH, as a marker of cell viability, may be biased by a direct inhibitory effect of ebselen or other chalcogenides on LDH, resulting in false protection in an in vitro system.     

Comparison of single pot and multiphase granulation. Part 2: Effect of the drying process on granules manufactured in a single pot granulator and dried either in situ or in a fluid bed dryer

The aim of this study was to highlight the effect of the drying process on granules manufactured in a pilot scale single pot granulator and dried either in situ or in a fluid bed dryer, for formulations differing in drug substance and its concentration (1%; 25%). Although most of raw data were within specifications, single pot drying tended to improve granule comprimability and seemed less sensitive to formulation. Moreover, it was demonstrated that the formulation impacted on granule median diameter, packing ability, comprimability, residual lower punch pressure and tablet dissolution kinetics. Interactions between process and formulation were highlighted concerning tablet tensile strength and uniformity of mass.     

Para-acyl-calix-arene based solid lipid nanoparticles (SLNs): a detailed study of preparation and stability parameters

The preparation and stability parameters of para-acyl-calix[4]arene based solid lipid nanoparticles (SLNs) have been investigated. Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) show a mean particle size of 130 nm. In terms of preparation parameters, using the solvent displacement method, the nature and the volume of the organic solvent, the concentration of the amphiphile and the presence of a co-surfactant in the organic phase have been shown to affect significantly the size of the produced SLNs. In contrast, the stirring speed, the viscosity and the acidity of the aqueous phase and the amphiphile hydrophobic chain length have been shown to have no effect. In terms of stability parameters, the ionic strength has been shown to affect the short-time SLN stability depending on both the anion and the cation studied, with sodium sulphate causing precipitation. Ultrasonic, ultraviolet or microwave treatments of the SLN suspensions have no effect on the size of the SLNs. The study of the effects of short time thermal treatment revealed that the SLNs are not affected by one freezing-defreezing cycle and are stable at 100 degrees C in suspension. It is difficult to reconstitute the SLN suspensions after freeze-drying. Finally, the temporal stability of these suspensions in water has been shown to be superior to 1 year. The long-term temporal stability of suspensions stored in saline solution has been investigated. It has been demonstrated that the most destabilising effects arise from the presence in the storage suspension of sulphate ions.1H NMR, X-ray powder diffraction (XPD) and AFM have also been carried out on the calix-arene based SLNs and demonstrate the presence of a semi-organised matrix structure for the SLNs.     

Effect of process and formulation variables on the preparation of parenteral paclitaxel-loaded biodegradable polymeric nanoparticles: A co-surfactant study

The purpose of this study was to evaluate the effect of process (homogenization speed and evaporation time) and formulation (aqueous/organic phase ratio, surfactant concentration, polymer type and concentration, and drug amount) variables on the preparation of paclitaxel-loaded biodegradable polymeric nanoparticles using modified solvent evaporation technique. Thereafter, a formulation was selected and subjected to evaluation of inclusion of a co-surfactant for further reduction of particle size. Particle size, encapsulation efficiency and in-vitro drug release kinetics were evaluated. It was observed that the inclusion of vitamin E TPGS (0.01%), Poloxamer 188 (0.5%) or Tween 80 (0.25%) reduced the particle size of nanoparticles to 230, 244 or 301 nm from 438 nm, respectively. Encapsulation efficiency increased for both vitamin E TPGS and Poloxamer 188 up to concentration at 0.010% and 0.25%, respectively, while this was not the case for Tween 80. Comparison of drug release kinetics demonstrated that drug release accelerated from paclitaxel-loaded biodegradable nanoparticles prepared with the inclusion of Tween 80 but was delayed for Poloxamer 188 and vitamin E TPGS. Thus, it was concluded that the particle size of the nanoparticles could be reduced further and the paclitaxel release kinetics could easily be adjusted by taking advantage by the inclusion of a co-surfactant.     

Dissolution stability studies of suspensions of prolonged-release diclofenac microcapsules prepared by the Wurster process: I. Eudragit-based formulation and possible drug-excipient interaction

The aim was to evaluate possible interaction in solid and liquid state of the drug with formulation excipients consequent to very fast drug release of diclofenac-Eudragit prolonged release microcapsules. The microcapsules were prepared by drug layering on calcium carbonate cores and coated with Eudragit RS 30D and L30D-55 as previously reported. Suspension of the microcapsules was prepared using microcrystalline cellulose/sodium carboxymethyl cellulose (Avicel CL-611) as medium. In vitro dissolution testing of the suspension was done, and, based on the dissolution results, possible interaction between diclofenac and Eudragit and Avicel in the medium was studied. Powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC) analyses were performed using 1:1 binary, 1:1:1 ternary mixtures and a ratio equivalent to that in the formulation. The mixtures were prepared by mixing the dispersions--Eudragit RS 30D or L30D-55 with the drug or other components, followed by drying at 60 degrees C for 48 h. Dry mixing was done using the powder equivalents of the polymers, Eudragit RS PO and L100-55, Avicel and calcium carbonate. In vitro dissolution of the suspended microcapsules showed a very fast release after 48 h (T50 = <1 h) compared to the solid microcapsules (T50 = 6 h). DSC curves of the formulation components or microcapsules did not show the characteristic endothermic peak of diclofenac at 287 degrees C. Powder X-ray diffraction of the binary or ternary mixtures of diclofenac and Eudragit polymers indicated reduction, shift or modification of the crystalline peaks of the drug or excipients at 2theta of 12 degrees and 18 degrees , suggestive of interaction. Some changes in drug peak characteristics at 18 degrees and 23 degrees were observed for Avicel/drug mixture, though not significant. The DSC curves of the binary mixture of diclofenac co-dried with liquid forms of Eudragit (i.e. RS 30D or L30D-55) revealed greater interaction compared to the curves of drug and powdered forms of Eudragit (RS PO or L100-55). This was depicted by greater shift in fusion points of the mixtures relative to the drug. However, comparing the RS and L-type Eudragit, the latter generally showed greater interaction with the drug. Interaction between diclofenac and L-type Eudragit polymers can occur in liquid formulations.