Solubility Behavior,Phase Transition,and Structure-Based Nucleation Inhibition of Etanidazole in Aqueous Solutions

Purpose. The solubility behavior, phase transition and inhibition of the nucleation process of etanidazole were characterized. Methods. Solubility measurements as a function of time permitted characterization of the solubility behavior and phase transition. The precipitate from saturated solutions was isolated and characterized by differential scanning calorimetry, polarized light microscopy, x-ray powder diffraction and coulometric analysis. The physical stability of metastable systems was examined in the presence of various structure-based nucleation inhibitors. Results. Etanidazole is soluble in water with an equilibrium solubility of 68.1 mg/mL, pH 6.5 with changes in pH having virtually no effect on the solubility. Etanidazole reaches concentrations in excess of 150 mg/mL within one hour. Etanidazole solutions prepared at 150 mg/ mL contained crystals after rotating for 24 hours. The crystals were isolated and characterized as etanidazole monohydrate. The solubility of etanidazole monohydrate in water increased with time reaching an equilibrium solubility of 68 mg/mL after 24 hours. Therefore, the solubility studies were actually determining the solubility of the more stable monohydrate form of etanidazole. Etanidazole solutions at concentrations of 50, 100 and 150 mg/mL were stabilized to varying degrees with structure-based nucleation inhibitors (imidazole, ethanolamine or diethanolamine). Conclusions. Anhydrous etanidazole undergoes a transition in aqueous solutions to the more stable monohydrate when the solubility of the monohydrate is exceeded. The physical stability of etanidazole solutions at 4°C is improved following autoclaving. The addition of structure-based nucleation inhibitors effectively stabilized the metastable systems.     

Stabilization of Lactate Dehydrogenase Following Freeze-Thawing and Vacuum-Drying in the Presence of Trehalose and Borate

Purpose. The purpose of this work was to investigate the effects of trehalose and trehalose/sodium tetraborate mixtures on the recovery of lactate dehydrogenase (LDH) activity following freeze-thawing and centrifugal vacuum-drying/rehydration. The storage stability of LDH under conditions of either high relative humidity or high temperature was also studied. Methods. LDH was prepared in buffered aqueous formulations containing trehalose alone and trehalose/'borate' mixtures. Enzymatic activity was measured immediately following freeze-thawing and vacuum-drying/rehydration processes, and also after vacuum-dried formulations were stored in either high humidity or high temperature environments. Also, glass transition temperatures (T_g) were measured for both freeze-dried and vacuum-dried formulations. Results. The T_gvalues of freeze-dried trehalose/borate mixtures are considerably higher than that of trehalose alone. Freezing and vacuum-drying LDH in the presence of 300 mM trehalose resulted in the recovery of 80% and 65% of the original activity, respectively. For vacuum-dried mixtures, boron concentrations below 1.2 mole boron/ mole trehalose had no effect on recovered LDH. After several weeks storage in either humid (100% relative humidity) or warm (45°C) environments, vacuum-dried formulations that included trehalose and borate showed greater enzymatic activities than those prepared with trehalose alone. We attribute this stability to the formation of a chemical complex between trehalose and borate. Conclusions. The high T_gvalues of trehalose/borate mixtures offer several advantages over the use of trehalose alone. Most notable is the storage stability under conditions of high temperature and high relative humidity. In these cases, formulations that contain trehalose and borate are superior to those containing trehalose alone. These results have practical implications for long-term storage of biological materials.     

Crystallization of Mannitol below Tg′ during Freeze-Drying in Binary and Ternary Aqueous Systems

Purpose. To characterize the phase transitions in a multicomponent system during the various stages of the freeze-drying process and to evaluate the crystallization behavior below Tg (glass transition temperature of maximally freeze-concentrated amorphous phase) in frozen aqueous solutions and during freeze-drying. Methods. X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study frozen aqueous solutions of mannitol with or without trehalose. By attaching a vacuum pump to the low-temperature stage of the diffractometer, it was possible to simulate the freeze-drying process in situ in the sample chamber of the XRD. This enabled real-time monitoring of the solid state of the solutes during the process. Results. In rapidly cooled aqueous solutions containing only mannitol (10% w/w), the solute was retained amorphous. Annealing of frozen solutions or primary drying, both below Tg, resulted in crystallization of mannitol hydrate. Similar effects were observed in the presence of trehalose (2% w/w). At higher concentrations (5% w/w) of this noncrystallizing sugar, annealing below Tg led to nucleation but not crystallization. However, during primary drying, crystallization of mannitol hydrate was observed. Conclusions. The combination of in situ XRD and DSC has given a unique insight into phase transitions during freeze-drying as a function of processing conditions and formulation variables. In the presence of trehalose, mannitol crystallization was inhibited in frozen solutions but not during primary drying.     

Molecular Mobility of Amorphous Pharmaceutical Solids Below Their Glass Transition Temperatures

Purpose. To measure the molecular mobility of amorphous pharmaceutical solids below their glass transition temperatures (Tg), using indomethacin, poly (vinyl pyrrolidone) (PVP) and sucrose as model compounds. Methods. Differential scanning calorimetry (DSC) was used to measure enthalpic relaxation of the amorphous samples after storage at temperatures 16-47 K below Tg for various time periods. The measured enthalpy changes were used to calculate molecular relaxation time parameters. Analogous changes in specimen dimensions were measured for PVP films using thermomechanical analysis. Results. For all the model materials it was necessary to cool to at least 50 K below the experimental Tg before the molecular motions detected by DSC could be considered to be negligible over the lifetime of a typical pharmaceutical product. In each case the temperature dependence of the molecular motions below Tg was less than that typically reported above Tg and was rapidly changing. Conclusions. In the temperature range studied the model amorphous solids were in a transition zone between regions of very high molecular mobility above Tg and very low molecular mobility much further below Tg. In general glassy pharmaceutical solids should be expected to experience significant molecular mobility at temperatures up to fifty degrees below their glass transition temperature.     

The Relationship Between Protein Aggregation and Molecular Mobility Below the Glass Transition Temperature of Lyophilized Formulations Containing a Monoclonal Antibody

Purpose. To find out if the physical instability of a lyophilized dosage form is related to molecular mobility below the glass transition temperature. Further, to explore if the stability data generated at temperatures below the glass transition temperature can be used to predict the stability of a lyophilized solid under recommended storage conditions. Methods. The temperature dependence of relaxation time constant, , was obtained for sucrose and trehalose formulations of the monoclonal antibody (5 mg protein/vial) from enthalpy relaxation studies using differential scanning calorimetry. The non-exponentiality parameter, , in the relaxation behavior was also obtained using dielectric relaxation spectroscopy. Results. For both sucrose and trehalose formulations, the variation in with temperature could be fitted Vogel-Tammann-Fulcher (VTF) equation. The two formulations exhibited difference sensitivities to temperature. Sucrose formulation was more fragile and exhibited a stronger non-Arrhenius behavior compared to trehalose formulation below glass transition. Both formulations exhibited <2% aggregation at t values <10, where t is the time of storage. Conclusions. Since the relaxation times for sucrose and trehalose formulations at 5°C are on the order of 10⁸ and 10⁶ hrs, it is likely that both formulations would undergo very little (<2%) aggregation in a practical time scale under refrigerated conditions.     

Crystallization Behavior of Mannitol in Frozen Aqueous Solutions

Purpose. To study the effect of cooling rate, the influence of phosphate buffers and polyvinylpyrrolidone (PVP) on the crystallization behavior of mannitol in frozen aqueous solutions. Methods. Low-temperature differential scanning calorimetry and powder X-ray diffractometry were used to characterize the frozen solutions. Results. Rapid cooling (20°C/min) inhibited mannitol crystallization, whereas at slower cooling rates (10°C and 5°C/min) partial crystallization was observed. The amorphous freeze-concentrate was characterized by two glass transitions at -32°C and -25°C. When the frozen solutions were heated past the two glass transition temperatures, the solute crystallized as mannitol hydrate. An increase in the concentration of PVP increased the induction time for the crystallization of mannitol hydrate. At concentrations of 100 mM, the buffer salts significantly inhibited mannitol crystallization. Conclusions. The crystallization behavior of mannitol in frozen solutions was influenced by the cooling rate and the presence of phosphate buffers and PVP.     

What is the True Solubility Advantage for Amorphous Pharmaceuticals?

Purpose. To evaluate the magnitude of the solubility advantage foramorphous pharmaceutical materials when compared to their crystallinecounterparts.Methods. The thermal properties of several drugs in their amorphousand crystalline states were determined using differential scanningcalorimetry. From these properties the solubility advantage for theamorphous form was predicted as a function of temperature using a simplethermodynamic analysis. These predictions were compared to theresults of experimental measurements of the aqueous solubilities of theamorphous and crystalline forms of the drugs at several temperatures.Results. By treating each amorphous drug as either an equilibriumsupercooled liquid or a pseudo-equilibrium glass, the solubilityadvantage compared to the most stable crystalline form was predicted to bebetween 10 and 1600 fold. The measured solubility advantage wasusually considerably less than this, and for one compound studied indetail its temperature dependence was also less than predicted. It wascalculated that even for partially amorphous materials the apparentsolubility enhancement (theoretical or measured) is likely to influencein-vitro and in-vivo dissolution behavior.Conclusions. Amorphous pharmaceuticals are markedly more solublethan their crystalline counterparts, however, their experimental solubility advantage is typically less than that predicted from simplethermodynamic considerations. This appears to be the result of difficulties indetermining the solubility of amorphous materials under trueequilibrium conditions. Simple thermodynamic predictions can provide a useful indication of the theoretical maximum solubility advantage foramorphous pharmaceuticals, which directly reflects the driving forcefor their initial dissolution.     

Optimization of Lyophilization Conditions for Recombinant Human Interleukin-2 by Dried-State Conformational Analysis Using Fourier-Transform Infrared Spectroscopy

Purpose. Examination of the dried-state conformation of interleukin-2 (IL-2) was used to determine the pH conditions and stabilizers that provide optimal storage stability for the lyophilized product. Methods. Fourier-transform infrared spectroscopy and accelerated stability studies which examined solubility, aggregate formation, and covalent cross-linking were used. Results. Varying the pH in the absence of excipients resulted in dramatic differences in the dried state conformation of IL-2. At pH 7, IL-2 unfolds extensively upon lyophilization while at pH below 5 it remains essentially native. Additional unfolding was observed upon incubation at elevated temperatures. A strong direct correlation between the retention of the native (aqueous) structure during freeze-drying and enhanced stability is demonstrated. IL-2 prepared at pH 5 is approximately an order of magnitude more stable than at pH 7 with regard to formation of soluble and insoluble aggregates. A similar pH profile was observed in the presence of excipients, although the excipients alter the overall stability profile. Additional accelerated stability studies examined the stabilizers necessary for optimal stability. Conclusions. Excipients with the capacity to substitute for water upon dehydration better preserve the native structure resulting in enhanced stability. Those that have high glass transition temperatures provide the highest level of stability during storage, although they do not prevent dehydration induced unfolding.     

Effects of Lyophilization on the Physical Characteristics and Chemical Stability of Amorphous Quinapril Hydrochloride

Purpose. To prepare amorphous quinapril hydrochloride(QHCl) by lyophilization and to compare its physical characteristics andchemical stability as a function of the initial pH of the pre-lyophilizedsolution. Methods. Amorphous QHCl samples were prepared bylyophilization from aqueous solutions. Solid-state characteristics wereevaluated by DSC, PXRD, and optical microscopy. Chemical degradation wasmonitored by an HPLC assay. Results. Amorphous QHCl samples obtained fromlyophilization exhibited variable glass transition temperatures, dependingon the pH and/or concentration of the starting aqueous solutions.Neutralized quinapril (Q) in the amorphous form, which has a T_gof 51°C, lower than that of its HCl salt (91°C), was significantlymore reactive than QHCl at the same temperature. The T_g oflyophilized samples prepared at various initial pH values correlated wellwith values predicted for mixtures of QHCl and Q. Their different reactionrates were related to their glass transition temperature, consistent withthe results from earlier studies obtained with amorphous samples made byprecipitation from an organic solution and grinding of the crystalsolvate. Conclusions. Lyophilization of different QHCl solutionsproduces mixtures of amorphous QHCl and its neutralized form Q, withT_g values intermediate to the values of QHCl and Q. As thefraction of Q increases the overall rate of chemical degradation increasesrelative to QHCl alone, primarily due to the increase in molecular mobilityinduced by the plasticizing effects of Q.     

Glucose-Binding Property of Pegylated Concanavalin a

Purpose. Concanavalin A (Con A) has been used in the development of sol-gel phase-reversible hydrogels for modulated insulin delivery. The usefulness of Con A has suffered from its poor aqueous solubility and stability. The goal of this study was to modify Con A with poly(ethylene glycol) (PEG) and examine the water solubility and stability of the PEGylated Con A. Methods. Con A was PEGylated using monomethoxy poly(ethylene glycol) p-nitrophenol carbonates, and the extent of PEGylation was determined by the fluorescamine method. The stability of the PEGylated Con A was examined by measuring the time-dependent absorbance at 630 nm. The binding affinities of glucose and allyl glucose to native- and PEGylated-Con A were measured by the equilibrium dialysis method. Results. The total number of PEG molecules that can be grafted to Con A was 10. As the number of grafted PEG chains per each Con A was increased up to 5, the binding affinity of glucose was gradually increased and reached the maximum. The solubility and stability of PEGylated Con A were improved significantly over those of native Con A. The binding affinity of allyl glucose to Con A was not changed much by PEGylation. When the extent of PEGylation was excessive (i.e., the number of grafted PEG chains per each Con A was larger than 5), however, the binding affinities of both glucose and allyl glucose were decreased significantly. Conclusions. PEGylation of Con A resulted in improved aqueous solubility and stability of Con A. The binding affinity of glucose increased and reached the maximum when the extent of PEGylation was 50%. Advantages of PEGylated Con A over native Con A are improved aqueous solubility, enhanced long-term stability, and higher glucose sensitivity.     

Characterisation of the Glass Transition of an Amorphous Drug Using Modulated DSC

Purpose. The use of modulated differential scanning calorimetry (MDSC) as a novel means of characterising the glass transition of amorphous drugs has been investigated, using the protease inhibitor saquinavir as a model compound. In particular, the effects of measuring variables (temperature cycling, scanning period, heating mode) have been examined. Methods. Saquinavir samples of known moisture content were examined using a TA Instruments 2920 MDSC at a heating rate of 2°C/min and an amplitude of ± 0.159°C with a period of 30 seconds. These conditions were used to examine the effects of cycling between - 50°C and 150°C. A range of periods between 20 and 50 seconds were then studied. Isothermal measurements were carried out between 85°C and 120°C using an amplitude of ± 0.159°C with a period of 30 seconds. Results. MDSC showed the glass transition of saquinavir (0.98 ± 0.05%w/w moisture content) in isolation from the relaxation endotherm to give an apparent glass transition temperature of 107.0° C ± 0.4C. Subsequent temperature cycling gave reproducible glass transition temperatures of approximately 105°C for both cooling and heating cycles. The enthalpic relaxation peak observed in the initial heating cycle had an additional contribution from a Tg 'shift' effect brought about by the difference in response to the glass transition of the total and reversing heat flow signals. Isothermal studies yield a glass transition at 105.9°C ± 0.1°C. Conclusions. MDSC has been shown to be capable of separating the glass transition of saquinavir from the relaxation endotherm, thereby facilitating measurement of this parameter without the need for temperature cycling. However, the Tg 'shift' effect and the number of modulations through the transition should be taken into account to avoid drawing erroneous conclusions from the experimental data. MDSC has been shown to be an effective method of characterising the glass transition of an amorphous drug, allowing the separate characterisation of the Tg and endothermic relaxation in the first heating cycle.     

Implications of Global and Local Mobility in Amorphous Sucrose and Trehalose as Determined by Differential Scanning Calorimetry

Purpose  To investigate the local and global mobility in amorphous sucrose and trehalose and their potential implications on physical stability. Methods  Amorphous sucrose was prepared by lyophilization while amorphous trehalose was prepared by dehydration of trehalose dihydrate. The variation in the effective activation energy of α-relaxation through glass transition has been determined by applying an isoconversional method. β-Relaxations were detected as shallow peaks, at temperatures below the glass transition temperature, caused by annealing glassy samples at different temperatures and subsequently heating at different rates in a differential scanning calorimeter. The effect of heating rate on the β-relaxation peak temperature formed the basis for the calculation of the activation energy. Results  α-Relaxations in glassy trehalose were characterized by larger activation energy barrier compared to sucrose, attributable to a more compact molecular structure of trehalose. The effect of temperature on viscous flow was greater in trehalose which can have implications on lyophile collapse. The size of the cooperatively rearranging regions was about the same for sucrose and trehalose suggesting similar dynamic heterogeneity at their respective glass transition temperatures. The activation energy of β-relaxations increased with annealing temperature due to increasing cooperative motions and the increase was larger in sucrose. The temperature at which β-relaxation was detected for a given annealing time was much less in sucrose implying that progression of local motions to cooperative motions occurred at lower temperatures in sucrose. Conclusions  Trehalose, having a lower free volume in the glassy state due to a more tightly packed molecular structure, is characterized by larger activation energies of α-relaxation and experiences a greater effect of temperature on the reduction in the activation energy barrier for viscous flow. The pronounced increase in cooperative motions in sucrose upon annealing at temperatures below (T _g −50) suggest that even a small excursion in temperature could result in a significant increase in mobility.     

Determination of the Viscosity of an Amorphous Drug Using Thermomechanical Analysis (TMA)

Purpose. To evaluate thermomechanical analysis (TMA) as a technique for determining the viscosity of amorphous pharmaceutical materials. This property of amorphous drugs and excipients is related to their average rate of molecular mobility and thus to their physical and chemical stability. Methods. Indomethacin was selected as a model amorphous drug whose viscosity has previously been reported in the literature. A Seiko TMA 120C thermomechanical analyzer was utilized in isothermal penetration mode to determine the viscosity of the amorphous drug over the maximum possible range of temperatures. Results. Using a cylindrical penetration geometry it was possible to accurately determine the viscosity of amorphous indomethacin samples by TMA over the temperature range from 35 to 75°C. The results were consistent with those reported in the literature using a controlled strain rheometer over the range 44–75°C. The limiting lower experimental temperature for the TMA technique was extended to significantly below the calorimetric glass transition temperature (T_g 42°C), thus allowing a direct experimental determination of the viscosity at T_g to be made. Conclusions. Thermomechanical analysis can be used to accurately determine the viscosity of amorphous pharmaceutical materials at temperatures near and above their calorimetric glass transition temperatures.     

Solubilization of Retinoids by Bile Salt/ Phospholipid Aggregates

Purpose. The capacity and specificity of bile salt (BS)/phosphatidylcholine (PC) mixed lipid aggregated systems in solubilizing four structurally related retinoids, etretinate, motretinid, fenretinide and N-ethyl retinamide, were determined. Methods. Excess solid drug was dispersed into sodium taurocholate (NaTC)/egg PC systems at lipid ratios of 10:0, 10:2 and 10 mM:10 mM in isotonic HEPES buffer, pH 6.5. A sensitive HPLC method was used to quantify the amount solubilized. The melting point and associated enthalpy change as well as the aqueous solubilities were also measured. Results. The retinoids had aqueous solubilities of less than 25 nM. The predicted aqueous solubility was less than 0.01 nM. The amount of retinoid in 10 mM NaTC was increased from three to four orders of magnitude relative to the aqueous solubility. Further increases in the amount solubilized were observed in the 10:10 mixed micelle dispersion. Fenretinide and N-ethyl retinamide were particularly well solubilized by BS and BS/PC aggregated systems which may be related to the presence of a cyclohexenyl ring. Conclusions. The discrepancy between the observed and predicted aqueous solubility may be due to self-association of the retinoids. Micellar/aqueous distribution ratios appear to be dominated by the hydrophobic effect, although specific interactions also are important. In considering intestinal absorption, the large increase in solubilization with BS/PC micelles would be capable of dramatically increasing the bioavailability in spite of the smaller effective diffusivity of the solubilized retinoid.     

Effect of Glass Transition Temperature on the Stability of Lyophilized Formulations Containing a Chimeric Therapeutic Monoclonal Antibody

Purpose. The purpose of this study is to highlight the importance of knowing the glass transition temperature, T_g, of a lyophilized amorphous solid composed primarily of a sugar and a protein in the interpretation of accelerated stability data. Methods. Glass transition temperatures were measured using DSC and dielectric relaxation spectroscopy. Aggregation of protein in the solid state was monitored using size-exclusion chromatography. Results. Sucrose formulation (T_g ~ 59°C) when stored at 60°C was found to undergo significant aggregation, while the trehalose formulation (T_g ~ 80°C) was stable at 60°C. The instability observed with sucrose formulation at 60°C can be attributed to its T_g (~59°C) being close to the testing temperature. Increase in the protein/sugar ratio was found to increase the T_gs of the formulations containing sucrose or trehalose, but to different degrees. Conclusions. Since the formulations exist in glassy state during their shelf-life, accelerated stability data generated in the glassy state (40°C) is perhaps a better predictor of the relative stability of formulations than the data generated at a higher temperature (60°C) where one formulation is in the glassy state while the other is near or above its T_g.     

Enthalpy Relaxation in Binary Amorphous Mixtures Containing Sucrose

Purpose. To compare the enthalpy relaxation of amorphous sucrose and co-lyophilized sucrose-additive mixtures near the calorimetric glass transition temperature, so as to measure the effects of additives on the molecular mobility of sucrose. Methods. Amorphous sucrose and sucrose-additive mixtures, containing poly(vinylpyrrolidone) (PVP), poly(vinylpyrrolidone-co-vinyl-acetate) (PVP/VA) dextran or trehalose, were prepared by lyophilization. Differential scanning calorimetry (DSC) was used to determine the area of the enthalpy recovery endotherm following aging times of up to 750 hours for the various systems. This technique was also used to compare the enthalpy relaxation of a physical mixture of amorphous sucrose and PVP. Results. Relative to sucrose alone, the enthalpy relaxation of co-lyophilized sucrose-additive mixtures was reduced when aged for the same length of time at a comparable degree of undercooling in the order: dextran PVP > PVP/VA > trehalose. Calculated estimates of the total enthalpy change required for sucrose and the mixtures to relax to an equilibrium supercooled liquid state (H) were essentially the same and were in agreement with enthalpy changes measured at longer aging times (750 hours). Conclusions. The observed decrease in the enthalpy relaxation of the mixtures relative to sucrose alone indicates that the mobility of sucrose is reduced by the presence of additives having a T_g that is greater than that of sucrose. Comparison with a physically mixed amorphous system revealed no such effects on sucrose. The formation of a molecular dispersion of sucrose with a second component, present at a level as low as 10%, thus reduces the mobility of sucrose below T_g, most likely due to the coupling of the molecular motions of sucrose to those of the additive through molecular interactions.     

Physical ageing and thermal analysis of PLGA microspheres encapsulating protein or DNA

PLGA microspheres undergo physical ageing but their ageing kinetics have not been reported, nor the effect of encapsulated protein or plasmid DNA on any associated changes to the glass transition. Differential scanning calorimetry (DSC) was used to measure the rate of ageing of various PLGA microsphere formulations, with temperature-modulated DSC used to accurately measure the associated glass transition. The Cowie-Ferguson model was applied to determine the parameters describing the enthalpy relaxation kinetics. We show that encapsulated proteins had no significant effect on the glass transition of the microspheres, whereas DNA and PVA were mild antiplasticising agents, particularly with high Mw PLGA. Physical ageing occurred through a range of enthalpy relaxation times (or modes) and was independent of both encapsulated protein and surfactant used during microsphere preparation. Analysis of accelerated ageing at 35 degrees C gave calculated enthalpy relaxation times to thermal equilibrium of 280-400 h. No ageing was observed < or = 10 degrees C and at 25 degrees C estimated relaxation times were at least one order of magnitude greater than at 35 degrees C. Ageing of PLGA microspheres therefore occurs at temperatures >10 degrees C, but relaxation will be far from equilibrium unless storage times and/or temperatures are prolonged or nearing the glass transition, respectively.     

Molecular Mobility and Fragility in Indomethacin: A Thermally Stimulated Depolarization Current Study

Purpose. To show that thermally stimulated depolarization currents (TSDC), which is a dielectric experimental technique relatively unknown in the pharmaceutical scientists community, is a powerful technique to study molecular mobility in pharmaceutical solids, below their glass transition temperature (T_g). Indomethacin (T_g = 42°C) is used as a model compound. Methods. TSDC is used to isolate the individual modes of motion present in indomethacin, in the temperature range between –165°C and +60°C. From the experimental output of the TSDC experiments, the kinetic parameters associated with the different relaxational modes of motion were obtained, which allowed a detailed characterization of the distribution of relaxation times of the complex relaxations observed in indomethacin. Results. Two different relaxational processes were detected and characterized: the glass transition relaxation, or -process, and a sub-T_g relaxation, or secondary process. The lower temperature secondary process presents a very low intensity, a very low activation energy, and a very low degree of cooperativity. The fragility index (Angell's scale) of indomethacin obtained from TSDC data is m = 64, which can be compared with other values reported in the literature and obtained from other experimental techniques. Conclusions. TSDC data indicate that indomethacin is a relatively strong glass former (fragility similar to glycerol but lower than sorbitol, trehalose, and sucrose). The high-resolution power of the TSDC technique is illustrated by the fact that it detected and characterized the secondary relaxation in indomethacin, which was not possible by other techniques.     

Potentiometric determination of ionisation constants for diphacinone and chlorophacinone in a dioxane–water cosolvent system

The purpose of this study was to determine the ionisation constants of two poorly soluble compounds, namely diphacinone and chlorophacinone, potentiometrically in 1,4-dioxane–water mixtures with ibuprofen used as a standard. In this study, Gran's method was employed for the calibration of glass electrode in cosolvent systems with pH measurements based on the concentration scale (p_cH). Aqueous pK_a values for the tested compounds were obtained by extrapolation on a Yasuda–Shedlovsky plot. It was demonstrated that the pK_a for ibuprofen determined using this method was consistent with those reported in literature. The technique was applied successfully to the two indandione derivatives, diphacinone and chlorophacinone. The present study demonstrated that the use of an organic cosolvent is effective in improving the solubility of compounds allowing potentiometric determination of ionisation constants that are otherwise difficult in aqueous solutions.     

Solvent Effects on the Solubility and Physical Stability of Human Insulin-Like Growth Factor I

Purpose. The solubility and physical stability of human Insulin-like Growth Factor I (hIGF-I) were studied in aqueous solutions with different excipients. Methods. The solubility of hIGF-I was determined by UV-absorption and quantification of light blocking particles. The physical stability of hIGF-I was studied with differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Results. Human IGF-I precipitated at low temperature in the presence of 140 mM benzyl alcohol and 145 mM sodium chloride. CD data showed that the tertiary structure of hIGF-I during these conditions was perturbed compared to that in 5 mM phosphate buffer. In the presence of benzyl alcohol 290 mM mannitol stabilized hIGF-I. Sodium chloride or mannitol by themselves had no effect on either the solubility or the tertiary structure. Benzyl alcohol was attracted to hIGF-I, whereas sodium chloride was preferentially excluded. The attraction of benzyl alcohol was reinforced by sodium chloride leading to salting-out of hIGF-I. The CD-data indicated interactions of benzyl alcohol with phenylalanine in hIGF-I. Thermal denaturation of hIGF-I occurred in all solutions with sodium chloride, whereas mannitol or benzyl alcohol had no effect on the thermal stability. The thermal stability of hlGF-I was thus decreased in 145 mM sodium chloride although it was excluded from hIGF-I. Conclusions. The self-association and thermal aggregation of hIGF-I is driven by hydrophobic interactions. Benzyl alcohol is attracted to hIGF-I and induces changes in the tertiary structure causing hydrophobic attraction of the protein at low temperatures.