LiquidâLiquid Phase Separation in Highly Supersaturated Aqueous

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Liquid-Liquid Phase Separation in Highly Supersaturated Aqueous Solutions of Poorly-Water Soluble Drugs – Implications for Solubility Enhancing Formulations Grace Ilevbare, and Lynne S. Taylor Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/cg301679h • Publication Date (Web): 18 Feb 2013 Downloaded from http://pubs.acs.org on February 23, 2013

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Crystal Growth & Design

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Liquid-Liquid Phase Separation in Highly Supersaturated Aqueous Solutions of Poorly-Water Soluble Drugs – Implications for Solubility Enhancing Formulations Grace A. Ilevbare, Lynne S. Taylor*

Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana, 47907.

*(L.S.T.) Address: Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, USA. Tel: +1-765-496-6614; fax: +1-765-494-6545; e-mail: [email protected]. ACS Paragon Plus Environment


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ABSTRACT Highly supersaturated aqueous drug solutions are often generated during drug testing and upon delivery to the patient. The phase behavior of such solutions appears complex and poorly understood, with the formation of drug aggregates and colloids often being reported. In this study, the phase behavior of eight hydrophobic poorly-water soluble drug molecules in highly supersaturated aqueous solutions was examined and colloid formation was explained in terms of liquid-liquid phase separation (LLPS). A relationship was found between the concentration at which LLPS was observed and the theoretically predicted amorphous “solubility” value, where the latter was predicted based on the thermodynamic properties of the crystalline solid/supercooled liquid and solution activity coefficients. A phase diagram for the ritonavir-water system as a function of temperature was used to demonstrate that LLPS occurs in the metastable region of the phase diagram and thus LLPS is a precursor to crystallization. Using an amorphous solid dispersion of ritonavir and poly(vinylpyrrolidone), it was shown that there is an upper limit to the extent of supersaturation achievable by a supersaturating dosage form and that this limit is dictated by the LLPS phase transition concentration. The approaches outlined in this study provide an alternative way to assess the properties of supersaturating systems, including the determination of the amorphous solubility.

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INTRODUCTION It is well know that certain drugs can form aggregates in aqueous solution. This phenomenon has been widely observed and investigated in the context of high throughput screening assays where non-specific interactions between drug aggregates and targets have been found to be an important mechanism leading to false positives, and this type of behavior has been termed promiscuous inhibition1-6. Aggregation of drugs is thought to inactivate the target by sequestration and partial denaturation of protein molecules, which are adsorbed to the surface of the aggregate. In a screen of nearly 300 randomly selected compounds, close to 20% of compounds showed behavior characteristic of promiscuous aggregation7, illustrating the apparent widespread tendency of drugs to form aggregates in aqueous milieu. Aggregation is typically observed when a solution of the drug solubilized in an organic solvent is diluted into an aqueous solution. The size of the aggregates thus formed has been reported to range from 50 nm to greater than 1000 nm with the size of the aggregates often being observed to change with time4-6. Although considered deleterious for drug high throughput screening assays, it has been hypothesized that formation of nano-sized aggregates below a certain critical size is responsible for the higher than anticipated bioavailability of certain very hydrophobic non-nucleoside reverse transcriptase inhibitors (NNRTIs)4. NNRTIs with good biological absorption were found to form aggregates with hydrodynamic radii of less than 100 nm and it was suggested that this may result in absorption directly into the lymphatic systemic via the microvilli cells in Peyer’s patches, found in the small intestine4. Colloid formation has also been noted for dyes8, as well as supersaturating bioactive delivery systems, consisting of an amorphous blend of the poorlywater soluble compound with a water soluble polymer9, 10. Although aggregation has been widely observed for chemically diverse organic compounds and is clearly important both from a fundamental and applied perspective, the underlying basis for the aggregation phenomenon is not well understood. It has been noted that aggregation occurs when a certain monomer concentration has been reached, and further addition of compound does not result in any further increase in monomer concentration1, 2. This implies that there is an upper limit of monomer concentration (i.e. free solute) that can be achieved for any given aggregate forming compound. This has important implications for any ACS Paragon Plus Environment


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situation where it is important to know the maximum achievable free solute concentration, e.g. in drug delivery for construction of dose–response curves, as well as when trying to maximize biological exposure. Unfortunately, the concentration at which aggregation occurs and hence, the maximum free solute concentration cannot currently be predicted since the thermodynamics of the drug aggregation process have not been established. However, reported observations that aggregate formation reproducibly occurs at a well-defined concentration, that the amount of aggregate increases when additional compound is added to the system while the free monomer concentration remains constant, and that aggregates are reduced by dilution, suggest that the underlying phenomenon resulting in drug aggregates is of liquid-liquid phase separation. Liquid-liquid phase separation (LLPS) is a well-known process in the study of polymer blends, protein solutions and organic crystallization and leads to a two phase system consisting of a solute rich phase and a solvent rich phase11-15. Liquid-liquid phase transitions have not been widely investigated in aqueous solutions of lipophilic drugs, which typically have extremely low concentrations (0.999) over the concentration range. The regression intercept for the calibration curve was very small and was not statistically significant compared to zero. The equilibrium solubility of clozapine, clotrimazole, loratadine, ketoconazole were been reported in ref. 16, while the equilibrium solubility of felodipine and ketoconazole were reported in ref. 17 and ref. 18, respectively. The equilibrium solubility of ritonavir in 500 µg/mL Tween 80 and 25 µg/mL poly(vinyl pyrrolidone) solution was also determined by HPLC using the above mentioned method.

Theoretical and Experimental Amorphous “Solubility” The theoretical amorphous solubility of the model compounds was estimated using the experimentally determined crystalline solubility, the free energy difference between crystalline and amorphous forms (∆→ ), as well as the activity of the amorphous solute saturated with water ( exp [ − I (a2 ) ]) :  ∆G  Camorphous = Ceq .exp [ − I (a2 )] .exp  a →c   RT  The [ − I (a2 )] term was derived by Murdande et al.19,

20

(1)

and is applied herein. The free energy change for

transformation of an amorphous supercooled liquid to a crystalline solid was estimated from the heat of fusion (∆Hf) and the melting temperature (Tm) of crystalline compound determined from DSC analysis, using the Hoffman equation21 which has been found previously to provide a good estimate of this quantity22:

∆Ga →c =

∆H f (Tm − T )T Tm2

(2)

The melting temperature, heat of fusion and crystallization tendency classification of clotrimazole, felodipine, indomethacin, ketoconazole, loratadine and ritonavir have been previously reported by Baird et al. in reference23. The methods described by Baird et al.23 and Van Eerdenburgh et al.24 were used to determine melting temperature, heat of fusion and the crystallization tendency classification of clozapine and efavirenz. The activity of the amorphous solute saturated with water was estimated by applying the Gibbs-Duhem equation to water sorption isotherm data for the amorphous material. The detailed thermodynamic analysis is ACS Paragon Plus Environment

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presented in ref. 19. The water sorption isotherm for amorphous ritonavir was determined using the method described in ref. 16. Amorphous samples of ritonavir were prepared by a solvent evaporation method, specifically spin-coating. Spin-coating was performed using KW-4A spin-coater (Chemat Technology, Inc., Northridge, CA). Crystalline ritonavir (30 mg) was dissolved in 0.5 mL of methanol. Two to three drops of solution were placed on a circular glass slide (18 mm diameter,VWR International, LLC (Radnor, PA)) and spin-coated. Residual solvents were removed by drying the amorphous films under vacuum at room temperature for ~24 hours. The glass slides were weighed before and after spin coating (after drying) to determine weight of the amorphous film. Prior to use, the films were analyzed by powder X-ray diffraction and cross-polarized light microscopy to verify their amorphous nature. The experimental amorphous “solubility” of ritonavir was determined by dissolving the amorphous film prepared using the aforementioned method in the dissolution medium of interest at a constant temperature of 37 o

C. Powdered amorphous ritonavir was not used because agglomeration occurs upon immediate contact with the

dissolution medium, resulting in a very slow dissolution rate. The glass slide was placed on a star-shaped stir bar (VWR International, LLC (Radnor, PA)) and the solution stirred at a speed of 300 rpm using a digital stir plate (Corning, PC 420D, Corning Inc., NY). Solution concentration was measured using a SI Photonics (Tuscon, Arizona) UV/Vis spectrometer coupled to a fiber optic probe (path-length 5mm). Wavelength scans (200 – 450 nm) were performed at 45 seconds time intervals. Second derivatives (SIMCA P+ V. 12 software (Umetrics Inc.,Umea Sweden) of the spectra were taken for the calibration and sample data in order to mitigate particle scattering effects. Calibration solutions were prepared in methanol.

Determination of Co-existence Boundary [CL –

L

Boundary (Liquid-Liquid Phase Transition

Concentration)] at Equilibrium Temperature as a Function of Concentration ACS Paragon Plus Environment


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Solubilized drug (4 mg/mL) in methanol was titrated into a temperature controlled solution, buffered (10 mL) at a pH where the compound of interest is un-ionized, using a syringe pump (Harvard Apparatus, Holliston, MA). The solution was stirred at a speed of 300 rpm. The CL – L boundary (also referred to as the liquid-liquid phase transition concentration) at an equilibrium temperature was determined as the concentration where an increase in intensity of light scattered from the drug solutions was observed. Light scattering was detected by monitoring the extinction at non-absorbing wavelengths ranging from 280 – 450 nm using a SI Photonics UV/Vis spectrometer (Tuscon, Arizona), fiber optically coupled with a dip probe (path-length 5 mm), while solubilized drug was continuously added to the solution using the syringe pump. Evolution of turbidity was characterized by an increase in the extinction between 280 – 450 nm. For ritonavir, the CL –

L

boundary in

double-distilled water was determined as a function of temperature (10 – 65 oC) and ionic strength. The pH of the solution before and after the experiment was >7.0; ritonavir is predominantly un-ionized at this pH (pKa values of 1.8 and 2.6

25

). In addition, the impact of the organic solvent used to solubilize the compound and

other pre-dissolved additives in solution on the CL –

L

of ritonavir was determined. Ritonavir was solublized

using two methods: dissolving pure drug in two different organic solvents (methanol and terahydrofuran (THF)) and dissolving pure drug under very acidic conditions (0.1M HCl). The dissolved additives evaluated used were poly(vinyl pyrrolidone) (PVP) and Tween 80 at concentrations of 25 µg/mL and 500 µg/mL, respectively. CL –

L

of ritonavir was confirmed using other spectroscopic techniques such as fluorescence and dynamic light

scattering as described below.

Verification of the Co-existence Concentration (CL –

L

Boundary) and Characterization of the

Dispersed Phase 0.5 mL of solubilized ritonavir (60 mg/mL) in methanol was added to double-distilled water (20 mL) at an equilibrium temperature to produce a solution with a concentration well above the LLPS concentration at the temperature of interest (10, 37, 50 oC). In all experiments, the resulting solution was very cloudy. The solution (containing the turbid phase) was characterized using cross polarized light microscopy (PLM). Sample slides ACS Paragon Plus Environment

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for PLM, were prepared by placing a small aliquot of the solution between a glass slide and a cover-slip. The cross polarized optical microscope used was a Nikon Eclipse E600 Pol microscope, with NIS-Elements version 2.3 software package (Nikon Co., Tokyo, Japan). The supernatant was then separated from disperse phase by ultracentrifugation at 40,000 RPM (equivalent of 274,356 x g) in an Optima L-100 XP ultracentrifuge equipped with Swinging-Bucket Rotor SW 41 Ti (Beckman Coulter, Inc., Brea, CA) for 15 minutes. Subsequently, the drug concentration in the supernatant was measured using a UV/Vis spectrometer (SI Photonics UV/Vis spectrometer (Tuscon, Arizona)). The water content in the pelleted phase was determined gravimetrically using a symmetrical gravimetric analyzer (SGA-100) (TA Instruments, New Castle, DE). Approximately 15 to 25 mg of sample was placed into the sample pan and dried at room temperature and very low relative humidity (< 5% RH) until the weight change was less than 0.01 wt% over 2 minutes. The percent weight loss was taken to represent the water content in the sample. The precipitated phase was further characterized using a differential scanning calorimeter (DSC). Thermal transitions were measured using TA Q2000 DSC (TA Instruments, New Castle, DE) attached to a refrigerated cooling accessory (RCS) (TA instruments, New Castle, DE). Both the DSC and RCS were purged with nitrogen gas. Tin was used for temperature calibration, while cell constant and enthalpy calibrations were performed using indium. Baseline calibration was performed by heating the empty cell from -50 to 300 oC at 20 o

C/min. The reference and sample pans were identical. The precipitate was sealed in an aluminum pan with a

pin hole in the lid. The thermogram was obtained by first cooling the sample, and then heating the sample at a rate of 20 oC/min in order to determine the glass transition temperature. The temperature range used was -25 – 150 oC. Thermal transitions were viewed and analyzed using the analysis software Universal Analysis 2000 for Windows 2000/XP provided with the instrument.

Fluorescence Spectroscopy



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A fluorescence probe technique was used to further evaluate the CL –

L

boundary of ritonavir at 37 oC.

Fluorescence spectra were obtained using a Fluromax-3 (Horiba Jobin Yvon, Edison, NJ) spectrometer. Pyrene, which exhibits different fluorescence characteristics depending upon the properties of the solubilizing medium26, 27, was used as a fluorescence probe. A known amount of pyrene solubilized in DMSO was added to distilled water to generate a pyrene concentration of 0.5 µg/mL. Ritonavir solubilized in methanol (4 mg/mL) was added to 1 mL distilled water (containing pyrene) in 5 µL increments and a measurement was taken after each addition. The fluorescence experiment was performed in the cuvette used for measurements. Emission spectra of pyrene were obtained by exciting the samples at 334 nm. Spectra were analyzed using GRAMS/AI V.7.02 software (Thermal Fisher Scientific, Inc., Waltham, MA). The LLPS concentration was characterized by a decrease in the ratio of intensity of the first (I1 at 373 nm) and the third peaks (I3 at 383 nm) of pyrene emission spectra; I1/I3 is a sensitive parameter characterizing the polarity of the probe’s environment26.

Dynamic Light Scattering (DLS) The CL – L boundary was verified by monitoring the scattering intensity and particle size of disperse phase in solution with time using dynamic light scattering (DLS). Simultaneous measurements were performed using the aforementioned UV/Vis spectrometer fiber-optically coupled with a dip-probe and the DLS instrument. DLS experiments were performed using a Nano-Zetasizer (Nano-ZS) from Malvern Instruments (Westborough, MA) and its software, dispersion technology software (DTS). A backscatter detector was used and the scattered light was detected at an angle of 173°. This optical configuration maximizes the detection of scattered light while maintaining signal quality. A quartz flow-through cuvette (Malvern Instruments (Westborough, MA)), coupled with a MasterFlex® Easy-Load® peristaltic pump (Cole Parmer, Vernon Hills, IL) was used for continuous sampling. The flow rate used was 1 mL/min. Measurements were made approximately every 7 minutes. The experimental conditions used to the determine CL –

L

boundary (characterized using the scattered intensity) are

described above.


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The size of the dispersed phase was determined using a supersaturated solution generated by a single addition of solubilized drug (4 mg/mL) to water (50 mL) to make an initial ritonavir concentration corresponding to the CL –

L

boundary (LLPS concentration) of ritonavir under the same conditions (water at 37 oC) . The DLS

experiment commenced immediately after the creation of the supersaturated solution and the size of the dispersed phase was monitored with time.

Determination of Co-existence Boundary [Binodal (TL-L Boundary)] and Spinodal (Tsp) as a Function of Temperature The method used in this study to determine the co-existence curve as a function of temperature, the TL-L boundary for phase separation, has been described previously described (Lafferrère et al.)12. The TL – L boundary was characterized by the intensity of light scattered from the drug (ritonavir) solutions. Light scattering intensity measurements were performed at a scattering angle of 90o ( I 90o ) by placing a light-emitting diode (474 nm) at a right-angle to the direction of detection. Scattered light was detected using the abovementioned SI Photonics UV/Vis spectrometer. Experiments were carried out in a 100 mL double jacketed glass vessel connected to a water bath (Julabo USA Inc., Allentown, PA). The temperature of the water bath was automatically controlled using the software (Julabo Easy Temp) provided with the instrument (Julabo USA Inc, Allentown, PA). Solubilized drug (4 mg/mL) in methanol was added to buffer solution (pH 6.8) to generate a known concentration. The temperature was initially fixed to be well above the co-existence curve, and the temperature was then slowly lowered at a rate of 0.5 oC/minute. The onset of phase separation into two liquid phases, typically referred to as the solution clouding temperature, Tcloud, was characterized by an increase in scattering intensity at 474 nm. To determine the temperature of solution clarification, the same experiment was repeated, but in the reverse temperature order. A solution of known concentration was generated and the temperature was initially fixed below the co-existence curve, so that the solution was cloudy at this temperature.

The

temperature of the solution was then slowly increased until the solution clarified. The minimum temperature at which the solution clarified is denoted by Tclarify. The average of Tcloud and Tclarify was taken as TL-L. ACS Paragon Plus Environment


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The location of the spinodal line was estimated from the measured temperature dependence of

I 90o , which is

associated with this LLPS (TL-L boundary). The TL-L boundary and light scattered intensity

( I ) was 90o

determined as described above. The spinodal temperature (Tsp) at a concentration, c, was estimated from linear extrapolation of the reciprocal intensity, temperature at which

I 90−1o ,c = 0 .

I 90−1o ,c , of the light scattered at 90o as a function of temperature to the

Examples of the type of linear extrapolation of

I 90−1o ,c performed herein was

presented by Lafferrère et al. in ref. 12. The mathematical basis for this estimation is presented in ref. 12 and ref. 28.

Dissolution of an Amorphous Solid Dispersion of Ritonavir An amorphous solid dispersion of ritonavir and PVP was prepared by the solvent evaporation method. A 1:9 (wt%) ratio of ritonavir to PVP was dissolved in 100% methanol. The solution was dried in a rotary evaporator apparatus (Brinkman Instruments, Westbury, NY), with the water bath at 65 oC. The sample was then placed under vacuum for 24 hours to remove residual solvent. Dissolution of the amorphous solid dispersion of ritonavir was performed in 100 mM sodium phosphate buffer at pH 6.8, 37 oC, under non-sink conditions. The solution was stirred at a speed of 300 rpm, using a stir bar and digital stir plate (Corning, PC 420D, Corning Inc., NY). The solid was added to 50 mL buffer solution in small increments of ~3 mg every 10 minutes until the maximum amorphous “solubility” of ritonavir was attained. Solution concentration and extinction measurements were performed using the abovementioned UV spectrometer coupled to a fiber optic probe. Data collection started after the initial addition of amorphous solid.


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RESULTS AND DISCUSSION

Characterization of Liquid-Liquid Phase Separated (LLPS) Solutions When a high concentration of ritonavir solubilized in a small volume of organic solvent was added to water, the resultant solution was observed to be cloudy with a slight bluish color. Examination of this system under cross polarized light, revealed the presence of small, non-birefringent, spherical droplets (Figure 1), indicating a second dispersed phase with a liquid-like structure, suggesting that liquid-liquid phase separation has occurred. To determine the concentration where the second phase formed and to confirm its liquid-like structure, a combination of techniques were used. Figure 2 shows a plot of extinction (at a wavelength of 280 nm, UV measurement) and mean count rate (in kilocounts per seconds (kcps), DLS measurement) as a function of concentration for ritonavir in water at 37 oC. Data was collected simultaneously using both instruments. It clear from the UV extinction data shown in Figure 2 that turbidity (indicative of the presence of a second scattering dispersed phase12, 16, 29) evolved at a certain drug concentration. At a similar concentration, the count rate as measured by DLS is observed to increase dramatically, confirming the presence of a second phase. This phase had an initial mean droplet size of approximately ~350 nm, which increased with time, consistent with coalescence of the droplets, commonly observed in LLPS (Figure 3)30. The changes were observed at ritonavir concentrations of 37.2 ± 0.9 µg/mL and 39.8 ± 0.3 µg/mL for the UV/Vis and DLS methods, respectively. At concentrations below the LLPS concentration (39.8 ± 0.3 µg/mL, in water), meaningful particle size data (using DLS) could not be obtained since formation of the dispersed phase starts at ~39.8 ± 0.3 µg/mL. Since neither the turbidity measurements nor the DLS data provide information about structure of the disperse phase, a fluorescence probe was used to investigate the local environments in the solution. Environmentally sensitive fluorescence probes have been widely used to study different phases in solution including micelles and membranes26, 27. If the disperse ritonavir phase formed has a liquid-like structure as suggested by Figure 1, then it would be expected that a hydrophobic probe molecule would partition into this phase (ritonavir has a Log P of 5.98

23

and thus is much more hydrophobic than water) and consequently undergo a change in emission

characteristics relative to the emission spectrum in water. In contrast, if the disperse phase is crystalline, no ACS Paragon Plus Environment


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change in probe emission spectrum would be expected since pyrene will not penetrate the crystal lattice. For pyrene, the I1/I3 emission peak ratio is known to be sensitive to the polarity of the probe environment26. The dependence of pyrene I1/I3 emission peak ratio on ritonavir concentration at 37 oC is illustrated in Figure 4. I1/I3 remains constant up to a concentration of 39.0 µg/mL and then decreases sharply at higher concentrations. A decrease of the values of I1/I3 indicates that pyrene is now in a more hydrophobic environment than water, consistent with the formation of a ritonavir-rich droplet phase into which the probe partitions. The concentration where the probe emission spectrum changes dramatically is very similar to the results from UV and DLS analysis. In summary, UV/Vis spectrometry, DLS and fluorescence methods indicate that a second phase of ritonavir forms when the concentration of ritonavir exceeds ~40.0 µg/mL in water at 37 oC with the environmentally sensitive fluorescent probe indicating that the second phase is a liquid phase. Considering that the equilibrium solubility of the stable polymorph of ritonavir (Form II) in water at 37 °C is approximately 2.4 ± 0.03 µg/mL and the solubility of the high energy polymorph (Form I) is only a factor of approximately 5 higher31, 32, it is clear that LLPS phase transition of this system leads to a solution that is metastable with respect to all of the crystalline forms. Furthermore, given that the experimental temperature is well below the melting point of ritonavir (121 °C) 32, 33, it is also clear that that the liquid phase must be a supercooled liquid or glassy form of ritonavir, most likely containing some water. The DSC thermogram of the isolated disperse phase (Figure 5) showed a thermal event characteristic of a glass transition (Tg) at 0.4 oC, confirming that it is a supercooled liquid at all the experimental temperatures utilized in this study. Exothermic peaks, characteristic crystal melting was absent. Since the Tg of pure amorphous ritonavir is 50.4 oC32, 33, the lower Tg observed in this study is consistent with a ritonavir-rich phase that contains a few percent of water since it is well known that water depresses the glass transition event34. The gravimetrically determined water content in the ritonavir-rich phase was around 4 – 5% (Table 1). This water content is comparable to the estimated water sorption of amorphous ritonavir (undercooled melt) at 100% RH, obtained by extrapolating the experimentally determined percent weight gain of ritonavir as a function RH at a constant temperature (Figure


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6) and would also be expected to reduce the Tg of ritonavir since water acts a plasticizer. The magnitude of Tg reduction is consistent with observations that Tg is reduced by about 10 °C for each 1% of water sorbed22, 34. The composition of the continuous phase was also determined, following its separation from the disperse phase, and was found to be a water-rich phase with a ritonavir concentration of 36.0 µg/mL. This concentration corresponds well to the concentration where the second phase starts to appear based on turbidity, DLS and fluorescence measurements’, suggesting that equilibration between the two phases is achieved very quickly during continuous solute addition. Based on the composition of the disperse phase, i.e. a highly ritonavir rich phase, it is expected that the phase boundary for the formation of this second phase should be related to the amorphous “solubility”, or in more rigorous terms, the difference in the thermodynamic activity of the crystalline solid and the water-saturated supercooled liquid (amorphous solid) phase of ritonavir. This indeed appears to be the case where the estimated (using Equation 1) and experimental amorphous “solubility” of ritonavir in water yielded values of 39.2 µg/mL and 34.2 µg/mL, respectively. A number of additional hydrophobic, poorly water soluble compounds were also evaluated; the physicochemical properties of the compounds investigated herein are summarized in Table 2. The concentration at which the liquid-liquid phase transition was observed for these model compounds is summarized in Table 3. The crystalline solubility and theoretical amorphous “solubility” of the compounds are also presented in Table 3. For all the compounds, there is a good agreement between the predicted values for the liquid-liquid phase boundary based (amorphous “solubility”) upon consideration of thermodynamic properties of the crystal and supercooled liquid phase, and the experimentally observed concentrations where a second phase was detected. Based on Equation 1, it is expected that the liquid-liquid phased boundary will vary from one compound to another since the compounds investigated herein have different thermodynamic properties (i.e. Tm and ∆Hf) and equilibrium solubility (Ceq) values. Based on the excellent agreement between the predicted amorphous “solubility” values and the experimentally determined onset of LLPS, it is apparent that the analytical approaches used herein provide an alternative way to experimentally determine the amorphous “solubility” of some types of compounds (i.e. those that undergo LLPS prior to crystallization) without the need to produce ACS Paragon Plus Environment


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and dissolve a reference amorphous material. All of the compounds investigated in this study can be classified as slow crystallizers23 which may explain their tendency to undergo LLPS prior to crystallization. To ensure that the observed LLPS phenomenon was not promoted by the addition of a small amount of methanol in which the drugs were solubilized, alternative organic solvents and pH lowering were used to solubilize certain compounds. Table 4 summarizes the LLPS concentration of ritonavir, efavirenz and loratadine determined by titrating pH-solubilized drug into buffer (pH 6.8). The concentration of the stock solution of pHsolubilized drug (concentration of drug in aqueous solution at pH 1.0) is also indicated in Table 4. The pH of the solutions after the experiment ranged from ~4.0 to 6.0; ritonavir (pKa 1.8 & 2.6 25), efavirenz (pKa 10.2 35) and loratadine (pKa 5.0

36

) were predominantly un-ionized during the experiment. Comparing the values in

Tables 3 and 4 (organic solvent and pH solubilized drug, respectively), it is clear that the LLPS concentration of these model compounds is comparable irrespective of the method by which the drug was solubilized. Table 5 shows that the type of organic solvent used for drug solubilization and the rate of titration (addition) of solubilized drug into aqueous solution also have minimal impact on the experimentally observed LLPS concentration. In general, the aforementioned experimental observations are consistent with several key characteristics of colloidal formation phenomena which have been widely reported for a number of compounds: (1) a second phase was not observed until the concentration of drug surpassed a threshold known as the “critical aggregation concentration” (CAC) whereby above the CAC, the number of aggregates increased linearly with added organic material1-4, (2) aggregates formed beyond the CAC are not crystalline in nature since sequestration of proteins, which was observed in the presence of these particle aggregates, is not characteristic of crystalline materials1, 7, 37

, (3) the concentration of monomeric organic molecules (free drug) in solution is constant above the CAC and

further addition of compound does not result in any further increase in monomer concentration1, 2, and (4) the CAC of an organic molecule in a given buffer is relatively constant2.


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Temperature-Concentration Phase Diagrams In order to further explore the LLPS behavior of ritonavir, clear and cloud point determinations were performed in order to establish the co-existence curve, also called the binodal, for the two liquid phases of ritonavir, as a function of temperature. The resultant phase diagram for ritonavir is shown in Figure 7; the equilibrium crystalline solubility, binodal (determined from clear and cloud point measurements) and spinodal curves (determined using the methods of Lafferrère et al.12) are shown. Also plotted in Figure 7 are the phase transition concentrations obtained using the continuous solvent addition method described above. Interestingly, the binodal curve [TL-L boundary (co-existence boundary as a function of temperature)] and the phase transition concentration are very similar, consistent with a low energy barrier to LLPS once the co-existence concentration is reached and exceeded. In other words, the continuous addition of solubilized drug with the concurrent monitoring of turbidity appears to give a reasonable estimate to the co-existence concentration whereby there is minimal supersaturation with respect to the co-existence concentration prior to the appearance of the second phase, in agreement with previous reports15,

16

. This is also consistent with the small concentration range

between the binodal and spinodal curves. It is useful to divide the phase diagram shown in Figure 7 into two regions in order to understand how the potential phase behavior of a supersaturated solution varies with concentration. In region I, the solution is supersaturated with respect to the crystalline solid and therefore crystal nucleation and growth can occur. Above the co-existence (binodal) curve, in region IIa, the solution is supersaturated both with respect to LLPS and crystal nucleation. LLPS will only occur if the kinetics of crystal nucleation is slow relative to the LLPS process. At 37 oC, under similar experimental conditions (34 µg/mL drug in water), ritonavir has a long induction time for crystal nucleation, >2 hours, thus LLPS is kinetically favored at high supersaturations relative to crystallization. In region IIb, above the spinodal curve, liquid-liquid demixing is spontaneous and occurs by the process of spinodal decomposition; between the binodal and spinodal curves, the process is activated and requires nucleation of the new liquid phase. It is clear from Figure 7 that the metastable zone for LLPS (distance between binodal and spinodal curves) is small, as observed



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previously12, and consistent with the agreement between the continuous solute addition method and the clear and cloud point measurements. Based on the aforementioned experimental observations, as well as theoretical thermodynamic concepts, it is clear that highly supersaturated solutions of ritonavir undergo LLPS, where the ‘liquid’ undergoing phase separation from the bulk solution phase is a water-saturated viscous supercooled liquid, and that this transition precedes crystallization, in other words, as highlighted by Bonnet et al.30, this system has a submerged liquidliquid phase within the metastable zone for crystal nucleation. The two phases – bulk solution phase and drugrich disperse phase – are in equilibrium, and there is a relationship between the liquid-liquid phase equilibrium (LLPE) and solid-liquid phase equilibrium (SLPE). Since this is liquid-liquid equilibrium, the activity of the solute in the bulk solution phase (LLPE) is equivalent to the activity of the water-saturated supercooled melt. The activity of both liquid phases is higher than the activity of the solid (crystalline) phase in equilibrium with the solvent (SLPE) by a factor that depends on the free energy difference between the crystal and the supercooled liquid at the temperature of interest, corrected for any decrease in the activity of the supercooled liquid arising due to mixing with water. A more detailed thermodynamic analysis is provided in the appendix.

Role of Hydrophobic Interactions and Temperature in LLPS All of the compounds shown in Table 3 are observed to undergo LLPS are hydrophobic molecules with Log P values ranging from 2.24 – 5.98

23

. It is therefore intuitive, that the LLPS behavior observed for these

compounds is driven by hydrophobic interactions. In other words, the limited miscibility in water is due to the inability of the hydrophobic molecules to form energetically favorable interactions with water, leading to aggregation of the molecules to form a drug-rich phase. In order to further investigate this supposition, the coexistence concentration for ritonavir was evaluated as a function of ionic strength using sodium phosphate buffer (pH 6.8). Increasing ionic strength will promote aggregation of molecules via hydrophobic interactions38, 39

and thus would be expected to decrease the co-existence concentration. Table 6 summarizes the LLPS

concentration measured for ritonavir in different media of varying ionic strength and the theoretical amorphous ACS Paragon Plus Environment

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“solubility” of ritonavir. From Table 6, it is clear that the LLPS concentration of ritonavir decreases with increasing salt concentration. At a similar equilibrium temperature, the highest LLPS concentration was observed when the aqueous medium was pure water, while the lowest LLPS concentration was observed in 500 mM sodium phosphate buffer. Figure 8 shows the LLPS concentration–temperature profile of ritonavir in water and 100 mM sodium phosphate buffer. For the temperature range investigated, the LLPS concentration of ritonavir was always higher in water compared to 100 mM sodium phosphate buffer, by a factor of approximately 2. The change in LLPS concentration may be attributed to the “salting-out” effect. Salt enhances hydrophobic interactions by making the solvent more polar38. When the solvent ionic strength is increased by the addition of salts, the hydrophobic drug molecules will associate with other drug molecules in order to minimize the area of contact with the solvent, and as a result both crystalline drug solubility and co-existence concentration in water decreases with increasing salt concentration. The measured amorphous “solubility” corresponds well to the LLPS concentration under similar ionic strength conditions. In addition, the LLPS concentration of ritonavir corresponds well to the estimated theoretical amorphous “solubility” when the equilibrium solubility of ritonavir at a similar ionic strength was used to estimate the theoretical amorphous “solubility”. These results highlight the influence of the medium on both the equilibrium crystal solubility (factor of ~2 change over the ionic strength range investigated) and the concentration where LLPS is observed and hence the need for consistency when performing experiments. Since LLPS is the concentration at which the drug exceeds its miscibility limit with water, it is of interest to examine the co-existence concentration as a function of temperature. Typically, miscibility increases with an increase in temperature12, although more complex relationships are also possible. Figure 9 shows the phase transition concentration of ritonavir and efavirenz as a function of temperature in water. With the exception of ritonavir, the phase transition concentration of the compounds investigated in this study increased with temperature over the temperature range studied, as exemplified by efavirenz. Thus most compounds show the expected increase of miscibility of the drug with the solvent with increasing temperature. The miscibility of two partially miscible liquids typically increases with temperature since the unfavorable enthalpic interactions ACS Paragon Plus Environment


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become less impactful at higher temperatures due to increased thermal fluctuations. However, ritonavir showed a much more complex profile with temperature where the liquid-liquid phase transition concentration deceased to a minimum at ~35 oC and then increased with increasing or decreasing temperature, showing a U-shaped profile (Figure 9). This type of concentration–temperature profile has been reported for some surfactants40-44, whereby the dependence of the critical micelle concentration (CMC) with temperature exhibits a similar Ushaped profile. For surfactants, the minimum in the CMC-temperature curve (Tmin) is typically between 20 – 40 o 41-44

C

and the Tmin of highly hydrophobic ritonavir (Log P value of 5.98

23

) falls within this range of

temperature. Micellization, which is a type of hydrophobic aggregation behavior, is highly influenced by temperature as hydrophobic and polar interactions change with temperature leading to complex changes in the entropy and enthalpy of the micellization process which depends on the chemical structure of the surfactant. We surmise that the U-shaped profile of the co-existence concentration of ritonavir with temperature may have its origin in a similar phenomenon.

LLPS in the Context of Solubility Enhancement The ramifications of LLPS are very important in the context of the utilization of supersaturating dosage forms to enhance drug delivery. Based on thermodynamic considerations, it is clear that there is an upper solution concentration of “free” drug (i.e. the thermodynamic activity reaches a maximum) that can be achieved in aqueous solution; exceeding this concentration will lead to the formation of a new drug-rich phase and the thermodynamic activity of the two solution remains constant. Hence the co-existence concentration dictates the maximum equilibrium concentration of drug that can be maintained in one phase solution. Therefore, supersaturating formulations that lead to solution concentrations higher than the co-existence concentration should undergo LLPS. In addition, given the link between the SLPE and the LLPE, if a solubilization strategy that increases the crystal solubility is employed, the co-existence concentration should also increase, so that the activity ratio between the crystal and the supercooled liquid phase remains the same, at least for the case where the solubilizing additive is not incorporated into either the crystal or the liquid phases. These suppositions were ACS Paragon Plus Environment

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tested by: (1) creating increasingly concentrated ritonavir solutions through the addition of a PVP-ritonavir amorphous solid dispersion, and (2) determining the LLPS concentration after increasing the crystal solubility through addition of a surfactant as described below. First, we tested the supposition that the LLPS of ritonavir can be generated following dissolution of an amorphous solid dispersion consisting of ritonavir and a polymer. PVP was selected as the matrix polymer due to its high aqueous solubility, facilitating rapid dissolution of the solid dispersion. For this experiment, it was important to determine if PVP had any impact on the crystalline solubility of ritonavir. The equilibrium solubility of crystalline ritonavir in the presence of 25 µg/mL PVP was comparable to the equilibrium solubility of ritonavir in the absence of PVP, as shown in Table 7, and was found to be unchanged. Next, the LLPS concentration in the presence of 25 µg/mL PVP was determined and was found to be the same as the LLPS concentration in the absence of an additive (Table 7) suggesting that PVP is not incorporated into the ritonavirrich phase. We then sequentially added small masses of a 90:10 wt. % PVP-ritonavir solid dispersion to a pH 6.8 buffered solution and monitored UV absorbance and extinction. Figure 10 shows the resultant extinction data. When sufficient solid dispersion had been added to yield a ritonavir concentration of ~20 µg/mL, there was a dramatic increase in extinction, consistent with the formation of a second phase. This concentration is the same as the LLPS concentration in the same medium (Table 7). The extinction increased further with addition of more solid dispersion. DLS data confirmed the formation of a colloidal phase which an average size of approximately ~325 nm. Thus it is clear that LLPS can be generated by dissolution of a solid dispersion, and for the PVP-ritonavir system, the presence of the polymer does not impact the either the SLPE or the LLPE concentrations. The next situation that was considered is when the additive employed increases the equilibrium solubility of the crystalline form, in other words alters the concentration of the SPLE. Here we examined the impact of the additive on the concentration where LLPS is observed relative to its effect on the equilibrium crystal solubility. Tween 80, at a concentration above its critical micelle concentration was used for this purpose. The equilibrium solubility of crystalline ritonavir in 500 µg/mL Tween 80 was found to be 3.5 ± 0.10 µg/mL at 37 oC and pH ACS Paragon Plus Environment


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6.8, which is a factor of ~3 higher than the equilibrium solubility of ritonavir in the absence of additive (Table 7). The LLPS concentration of ritonavir was also found to increase in 500 µg/mL Tween 80 solution, to 46.1 ± 0.92 µg/mL. This value is also a ~3 times higher than the LLPS concentration of ritonavir in 100 mM sodium phosphate buffer solution in the absence of additive. These results shows that increasing the equilibrium solubility of the crystalline form results in a corresponding increase in the LLPS concentration, whereby the ratio of the LLPS concentration to the SLPE concentration remains constant. This can be rationalized based on the following considerations. For the SLPE, the thermodynamic activity of the solution saturated with respect to the crystalline phase is the same in the presence and the absence of the additive since the solid phase is identical in both instances. Likewise, for the LLPE, as long as the additive is not incorporated into the drug-rich phase, the activity of the drug-rich phase will be the same in the presence and the absence of the additive. Assuming that the activity coefficient for the drug in the bulk solution phase is constant for the concentration ranges studied (a reasonable assumption given the dilute solutions investigated in this study), then the ratio of the LLPS concentration to the equilibrium crystalline solubility must therefore be the same in the presence and absence of the additive. In other words, the maximum extent of supersaturation achievable in the presence of a solubilizing additive is not increased and the upper limit of the solution thermodynamic activity remains the same, even though a higher concentration of drug is in solution.

CONCLUSION In this study, the phase behavior of several pharmaceutically relevant, poorly-water compounds was investigated and the compounds were found to undergo LLPS in highly supersaturated solutions. The concentration at which LLPS was observed (that is the upper limit of “free” or monomeric drug) was comparable to the theoretical amorphous “solubility” of the various compounds. Phase diagrams for the ritonavir-water system indicated that LLPS occurs in a metastable or unstable region of the phase diagram and is thus a precursor to crystallization. The composition of the disperse phase in ritonavir-water solutions was confirmed to be a non-crystalline, ritonavir-rich phase composed of approximately 95% ritonavir. LLPS was ACS Paragon Plus Environment

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also observed during the dissolution of a supersaturating dosage formulation (ritonavir-PVP amorphous solid dispersion). In the presence of a surfactant, both the crystalline solubility and the LLPS concentration increased such that the ratio remained the same in the presence and absence of the solubilizing additive. The insights gained from this study have important implications for drug delivery where absorption and bioavailability of lipophilic compounds with low aqueous solubility are impacted by the extent and duration of supersaturation.



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APPENDIX

Thermodynamic Analysis – Connecting the Solubility of Water Saturated Amorphous Material to Liquid-Liquid Phase Separation In considering the solubility of an amorphous material, it is first necessary to briefly consider crystal solubility. The solubility of the crystalline solid is the concentration in solution following attainment of equilibrium between the crystalline solid and the solution phase whereby the solvent is not present in the crystalline phase and is given by45, 46:

ln xCL = − 1

where

xCL

1

∆Ga →c − ln γ L1 RT

(1)

is the mole fraction solubility of the crystalline material in the liquid of interest (L1, in this case

∆Ga→c the free energy difference between crystalline and

water), γ is the solution activity coefficient,

amorphous material at the temperature of interest, R is the gas constant and T is temperature of interest. Various approaches to estimate of the solubility of an amorphous material have been presented in the literature19-20, 47- 49. The simplest approach is to assume that the amorphous material is a supercooled liquid (SL) at the temperature of interest (as is true for the ritonavir system described in detail herein). Amorphous materials have no crystal lattice energy, so the ∆Ga →c term in Equation 3 is zero. For an amorphous material, Equation 3 reduces to:

ln xSLL = − ln γ L1 1

(4)

If it is assumed that the activity coefficient is a constant over the range of concentrations encompassing the crystalline solubility and amorphous solubility, which is a reasonable assumption for the poorly-water soluble compounds under consideration, then to a first approximation, Equations 3 and 4 can be used to estimate the amorphous (supercooled liquid) solubility by:

ln xSLL = ln xCL + 1

1

∆Ga →c RT


(5)

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In turn,

∆Ga →c at the temperature of interest can be estimated using various approaches (e.g. Hoffman

approach, heat capacity correction of thermodynamic quantities) which involve determining the enthalpy of fusion ( ∆H f ) at the melting point (Tm ) and using this value to estimate the free energy of fusion at the temperature of interest (T ) . Using the Hoffman equation21, which makes the assumption that the enthalpy of the supercooled liquid and that of the crystal vary linearly with temperature, i.e. that the heat capacity difference between the two is constant, ∆Ga →c can be estimated from the heat of fusion and melting point:

∆Ga →c =

∆H f (Tm − T )T Tm2

(6)

However, Equation 6 neglects the well-known absorption of moisture into an amorphous material that would be expected to occur as the material reaches equilibrium with the bulk solution phase. At equilibrium, the activity of a particular species,

i , in a multi-component system is identical in all phases at equilibrium: ai(1) = ai(2) = ai(3) = ... = ai( N )

(7)

The activity can be linked to the concentration in a given phase by:

ai1 = xiγ i1

(8)

where (γ i1 ) is the activity coefficient of the component in that phase. When the amorphous material (considered to be equivalent to a supercooled liquid) forms an equilibrium with the solution, the amorphous material will dissolve into the bulk liquid phase, and water will penetrate into the amorphous material. Using Equation 7 and 8, the phase equilibrium between the supercooled liquid and the bulk liquid solution can be written as follows:

aSLL = aSLL 1

(9)

2

xSLL γ SLL = xSLL γ SLL 1

1

2

2


(10)


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where

aSLL ( xSLL γ SLL ) is the activity of the compound in the bulk liquid solution and aSLL ( xSLL γ SLL ) is 1

1

1

2

2

2

the activity of the supercooled liquid. For an amorphous sample, xSLL ≠ 1 and/or γ SLL ≠ 1 ; in general, the 2

2

thermodynamic activity will not be unity as sorption of water by amorphous material reduces the thermodynamic activity. Thus, a reduction in the activity of the amorphous material following equilibration with water decreases xSLL2 . This is true for the case of ritonavir. Therefore a correction needs to be made to Equation 5 in order to account for moisture effects on the activity of the amorphous material. This correction has been elegantly documented by Murdande et al.19, 20 and their method was applied in this study to determine the impact of water absorbed into the amorphous material on its thermodynamic activity and hence estimated solubility advantage. This method involves determining the number of moles of water absorbed per mole of solute as a function relative humidity and estimating the water content at a relative humidity of 100 (water activity of 1). The activity of the amorphous solute

( xSLL γ SLL ) 2

2

is estimated by applying the Gibbs-Duhem

equation to water sorption isotherm data for the amorphous solid. The detailed thermodynamic analysis is presented in ref.19. Therefore, for an amorphous solid after incorporating the correction factor, Equation 4 and 5 can be rewritten as Equation 11 and 12, respectively:

(

ln xSLL = − ln γ L1 + ln xSLL γ SLL 1

ln xSL L = ln xCL + 1

1

2

2

)

(11)

∆Ga →c + ln xSLL γ SLL 2 2 RT

(

)

(12)

It is apparent from the above discussion that the amorphous solubility is linked to the crystalline solubility by Equation 12 and therefore can be predicted. Equation 2 (main text) and Equation 12 above are equivalent;

(

)

− I (a2 ) (Equation 2, values are listed in Table 2) is the same as ln xSLL γ SLL (Equation 12). 2 2

However, is the

amorphous solubility the same as liquid-liquid phase separation based on thermodynamic considerations? In order to address this, we will briefly review the thermodynamics of two partially miscible liquids. ACS Paragon Plus Environment

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Based on Equation 7 and 8, the phase equilibrium for two partially miscible liquids can be expressed as:

xL1 γ L1 = xL2 γ L2

(13)

where liquid phases 1 and phase 2 represents the bulk liquid solution and the drug-rich dispersed liquid phase, respectively. It is immediately apparent that the terms on the left hand side of Equation 13 are identical to the terms on the left hand side of the Equation 10, and thus a supercooled liquid (amorphous material) saturated with water, has the same thermodynamic activity as the same liquid phase formed through a process of liquidliquid phase separation and in equilibrium with the bulk solution. The amorphous solubility thus translates to the mole fraction of the solute in the continuous phase which is determined by the activity of the drug-rich phase and the activity coefficient of the drug in the continuous phase. In other words, the amorphous solubility can be approached by equilibrating an amorphous solid with the aqueous phase, the approach described by Murdande et al.19 in considerable detail, or by supersaturating the solution phase with solute so that the amorphous solubility is exceeded and a drug-rich amorphous phase is produced via liquid-liquid phase separation, as demonstrated herein.

ACKNOWLEDGEMENTS The authors thank AstraZeneca for their kind donation of felodipine. Support of the National Science Foundation through grant DMR-0804609 is gratefully acknowledged.



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FIGURES

10 µm Figure 1. Cross polarized light micrograph of ritonavir dispersed phase.

Figure 2. Simultaneous determination of CL-L boundary (LLPS concentration) of ritonavir in water at 37 oC using a UV/Vis spectrometer and dynamic light scattering instrument. In both cases, the concentration corresponding to the intersection of the dash-line is the LLPS concentration.


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Figure 3. Plot of mean dispersed droplet size versus time. The droplet size increased with time, consistent with coalescence of the droplets.

Figure 4. Ratio of intensity of the first (I1 at 373 nm) and the third peaks (I3 at 383 nm) of pyrene emission spectra as a function of concentration for ritonavir at an equilibrium temperature of 37 oC. A significant decrease in I1/I3 occurred at the concentration corresponding to the LLPS concentration (intersection of the red dashed-lines).



Page 30 of 42

30 -0.2

-0.4

Heat Flow (W/g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

-2.74°C 0.42°C(I)

-0.6

4.29°C

-0.8

-1.0 -15

Exo Up

-10

-5

0

5

10

Temperature (°C)

15 Universal V4.1D20 TA Instruments

Figure 5. Differential scanning calorimetry thermogram of ritonavir dispersed phase.

Figure 6. Moisture sorption isotherm of amorphous ritonavir at 37 oC. Estimated percent weight gain at 100% RH is 5.21% (extrapolation of the line to 100% RH). This value is comparable to the water content in the amorphous precipitate at 37 oC (Table 1).


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Figure 7. Phase diagram of ritonavir: equilibrium solubility, LLPS concentration, binodal and spinodal curves.

Figure 8. CL-L boundary (LLPS concentration) of ritonavir as a function of temperature in water (black squares) and 100 mM buffer (red circles).



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Figure 9. LLPS concentration of ritonavir (black squares) and efavirenz (red circles) as a function of temperature in distilled water.

Figure 10. Plot of extinction vs. time during dissolution of an amorphous solid dispersion of ritonavir and PVP (1:9 wt. % ratio, 100 mM sodium phosphate buffer, pH 6.8 and 37 oC). Approximately 3 mg of solid was added to buffer solution at the beginning of the experiment and subsequently every 10 minutes (red circles). The measured solution concentration of ritonavir at points C1, C2, C3 and C4 are 6.4, 11.8, 18.3 and 18.0 µg/mL based on UV absorption measurements. The theoretical concentrations based on mass of ritonavir added are 6.0, 12.0, 18.0 and 24.0 µg/mL at points C1, C2, C3 and C4, respectively. Extinction, which is indicative of LLPS, increased at the maximum (18.3 µg/mL) concentration. ACS Paragon Plus Environment

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TABLES

Table 1. Composition of drug-rich phase and concentration of drug in supernatant Temperature

Precipitate

Supernatant

(oC)

Drug

Water

(µg/mL)

10

95.97 %

4.03 ± 0.53 %

57.1 ± 1.09

37

95.13 %

4.87 ± 1.20 %

36.0 ± 0.49

50

94.85 %

5.15 ± 2.12 %

38.9 ± 0.35

n = 3; errors indicate one standard deviation.

Table 2. Physicochemical properties of the model compounds Tg (oC) b

% Moisture c

Exp [-I (a2)] d

121

Enthalpy of Fusion (kJ mol-1) 60.4

50

4.97

0.27

4.7

139

14.5

38

0.71

0.44

382.9

4.1

136

25.5

40

2.98

0.60

Ketoconazolea

531.4

3.5

150

52.6

45

4.30

0.28

Indomethacina

357.8

3.6

161

37.6

45

2.30

0.73

Felodipinea

384.3

2.2

147

30.9

45

0.79

0.85

Clotrimazolea

344.8

5.2

145

33.3

30

1.24

0.83

Clozapine

326.8

3.4

187

34.1

65

1.78

0.80

Compound

MW (gmol-1)

Log P

Melting Point (oC)

Ritonavir

720.9

5.6

Efavirenz

315.7

Loratadine

n = 3; errors indicate one standard deviation. a

Values [Log P, melting point (Tm), enthalpy of fusion (∆Hf) and Tg] reported in Baird et al., ref 23.

b

Glass transition temperature.

c

% Moisture gain at 95% relative humidity.

d

Activity of supercooled liquid (amorphous material).



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Table 3. Equilibrium solubility and LLPS concentration for model compounds at 37 oC Compound

pH of

Equilibrium

Predicted

LLPS

LLPS

Medium a

Solubility

amorphous

(µg/mL)

solubility w/water

(UV

(DLS

saturation

Detection)

Detection)

(µg/mL)

(µg/mL)

(µg/mL)

Concentration Concentration

Ritonavir

6.8

1.3 ± 0.20

20.6 ± 0.3

18.8 ± 0.07

18.6 ± 0.1

Ritonavir (in H2O)

7.4

2.4 ± 0.03

39.2 ± 0.6

37.2 ± 0.9

39.8 ± 0.3

Efavirenz

6.8

8.2 ± 0.20

19.8 ± 0.7

18.4 ± 0.8

17.0 ± 0.03

Loratadine

6.8

1.6 ± 0.10b

6.8 ± 0.4

7.6 ± 0.1

7.5 ± 0.4

56.6 ± 1.5

54.4 ± 0.5

54.5 ± 0.7

b

Ketoconazole

10.0

3.7 ± 0.10

Indomethacin

2.0

3.0d

26.7 ± 1.0

30.4 ± 0.5

31.8 ± 0.8

Felodipine

6.8

0.94 ± 0.08s

8.5 ± 0.2

9.8 ± 0.4

9.4 ± 0.4

Clotrimazole

10.0

0.4 ± 0.02b

4.0 ± 0.2

5.2 ± 0.1

5.0 ± 0.5

Clozapine

10.0

8.8 ± 0.10b

133.3 ± 1.8

135.8 ± 0.6

136.9 ± 1.7

a

Medium: 100 mM phosphate buffer, excluding ritonavir in H2O.

b

Equilibrium solubility values from Hsieh et al. (ref. 16).

c

Equilibrium solubility value from Konno et al. (ref. 17).

d

Equilibrium solubility value from Alonzo et al. (ref. 18).


Table 4. LLPS concentration of ritonavir, efavirenz and loratadine at 37 oC. The drug compounds were solubilized by in 0.1M HCl solution

Compound Concentration of pH-solubilized

Rate of addition

LLPS Concentration

(mL/min)

(UV Detection)

drug (µg/mL)

(µg/mL)

Ritonavir

300

0.100

18.8 ± 0.8

Efavirenz

240

0.125

16.9 ± 0.9

Loratadine

260

0.120

7.6 ± 0.2



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Table 5. Effect of organic solvent, ionic strength and rate of addition of solubilized ritonavir in organic solution on turbidity results in water at 37 oC

Organic

Dissolution

Rate of

Solvent

Medium

Addition

which Turbidity

Amorphous

(mL/min)

was first

“solubility”

detected (µg/mL)

(µg/mL)

0.01

35.0

34.2 ± 2.88

0.02

34.0

0.05

35.0

0.05

37.5

Ethanol

THF

Water

Water

Concentration at Experimental


Table 6. Effect of buffer ionic strength on LLPS concentration of ritonavir at 37 oC Dissolution Medium

Experimental Concentration at which

Equilibrium

Theoretical

Amorphous

turbidity was observed

Solubility

amorphous

“solubility”

(solvent switch

(µg/mL)

“solubility”

(µg/mL)

method) (µg/mL)

Distilled Water

34.2 ± 2.88

37.2 ± 0.90

2.4 ± 0.03

39. 2 ± 0.6

100mM Buffer

21.8 ± 1.86

18.8 ± 0.07

1.3 ± 0.20

20.6 ± 0.3

200mM Buffer

13.8 ± 1.97

14.3 ± 0.10

0.95 ± 0.11

15.3 ± 0.3

500mM Buffer

5.2 ± 0.65

4.7 ± 0.50

-

-



(µg/mL)


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Table 7. Effect of additive on LLPS concentration of ritonavir (RTV) at 37 oC using UV Method Compound

RTV-PVPa

Equilibrium

Equilibrium

LLPS

LLPS

Solubility in the

Solubility in the

concentration

concentration in

absence of

presence of

in the absence

the presence of

additive

additive

of additive

additive

1.3 ± 0.20 c

1.3 ± 0.03

RTV-Tween 80b

18.8 ± 0.07

3.5 ± 0.10 (2.7) d

18.3 ± 0.26 46.1 ± 0.92 (2.6) e

Concentration units in µg/mL. n = 3; errors indicate one standard deviation. a

Concentration of PVP in solution 25 µg/mL.

b

Concentration of Tween 80 in solution 500 µg/mL. CMC = 23.6 µg/mL or 0.018 mM.

c

Equilibrium solubility in 100 mM sodium phosphate buffer.

d

Ratio of equilibrium solubility in the presence to absence of additives is indicated in parentheses.

e

Ratio of LLPS concentration in the presence to absence of additives is indicated in parentheses.


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REFERENCES 1. Coan, K. E. D.; Shoichet, B. K. Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors. J. Am. Chem. Soc., 2008, 130, 9606 – 9612. 2. Coan, K. E. D.; Maltby, D. A.; Burlingame, A. L.; Shoichet, B. K. Promiscuous aggregate-based inhibitors promote enzyme unfolding. J. Med. Chem., 2009, 52, 2067 – 2075. 3. Feng, B. Y.; Simeonov, A.; Jadhav, A.; Babaoglu, K.; Inglese, J.; Shoichet, B. K.; Austin, C. P. A highthroughput screen for aggregation-based inhibition in a large compound library. J. Med. Chem., 2007, 50, 2385 – 2390. 4. Frenkel, Y. V.; Clark, A. D.; Das, K.; Wang, Y-H.; Lewi, P. J.; Janssen, P. A. J.; Arnold, E. Concentration and pH dependent aggregation of hydrophobic drug molecules and relevance to oral bioavailability. J. Med. Chem., 2005, 48, 1974 – 1983. 5. Pacheco, L. F.; Carmona-Ribeiro, A. M. Effects of synthetic lipids on solubilization and colloid stability of hydrophobic drugs. J. Colloid Interf. Sci., 2003, 258, 146 – 154. 6. Seidler, J.; McGovern, S. L.; Doman, T. N.; Shoichet, B. K. Identification and prediction of promiscuous aggregating inhibitors among known drugs. J. Med. Chem., 2003, 46, 4477 – 4486. 7. Feng, B. Y.; Shelat, A.; Doman, T. N.; Guy, R. K.; Shoichet, B. K. High-throughput assays for promiscuous inhibitors. Nat. Chem. Biol., 2005, 3, 146 – 148. 8. Brick, M. C.; Palmer, H. J.; Whitesides, T. H. Formation of colloidal dispersions of organic materials in aqueous media by solvent shifting. Langmuir, 2003, 19, 6367 – 6380. 9. Aisha, A. F. A.; Ismail, Z.; Abu-Salah, K. M.; Majid, A. M. S. A. Solid dispersions of α-Mangostin Improve its aqueous solubility through self-assembly of nanomicelles. J. Pharm. Sci., 2011, 1 – 11. 10. Tachibana, T.; Nakamura, A. A method for preparing an aqueous colloidal dispersions of organic materials by using water-soluble polymers: Dispersion of β-carotene by polyvinylprrolidone. Kolloid Z. Z. Polym., 1965, 203, 130 – 133.



Page 38 of 42

38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11. Tung, H-H.; Paul, E. L.; Midler, M.; McCauley, J. A. Critical Issues in Crystallization Practice. Crystallization of Organic Compounds: John Wiley & Sons, Inc.; 2008. 12. Lafferrère, L.; Hoff, C.; Veesler, S. Study of liquid-liquid demixing form drug solution. J. Cryst. Growth, 2004, 269, 550 – 557. 13. Veesler S.; Lafferrère, L.; Garcia, E.; Hoff, C. Phase Transitions in Supersaturated Drug Solution. Org. Process Res. Dev., 2003, 7, 983 – 989. 14. Groen, H.; Roberts, K. J. Nucleation, growth and pseudo-polymorphic behavior of citric acid as monitored in situ by attenuated total reflection fourier transform infrared spectroscopy. J. Phys. Chem. B, 2001, 105, 10723 – 10730. 15. Maeda, K.; Aoyama, Y.; Fukui, K.; Hirota, S. Novel Phenomenon of crystallization an emulsification of hydrophobic solute in aqueous solution. J. Colloid Interf. Sci., 2001, 234, 217 – 222. 16. Hsieh, Y.; Ilevbare, G. A.; Van Eerdenbrugh, B.; Box, K.; Sanchez-Felix, M.; Taylor, L. S. pH-induced precipitation behavior of weakly basic compounds: Determination of extent and duration of supersaturation using potentiometric titration and correlation to solid state properties. Pharm. Res. 2012, 20, 2738 – 2753. 17. Konno, H.; Handa, T.; Alonzo, D. E.; Taylor, L.S. Effect of polymer type on the dissolution profile of amorphous solid dispersions containing felodipine. Eur. J. Pharm. Biopharm., 2008, 20, 493 – 499. 18. Alonzo, D.; Zhang, G.; Zhou, D.; Gao, Y.; Taylor, L. S. Understanding the behavior of amorphous pharmaceutical systems during dissolution. Pharm. Res., 2010, 27, 608 – 618. 19. Murdande, S. B.; Pikal, M. J.; Shanker, R. M.; Bogner, R. H. Solubility advantage of amorphous pharmaceuticals I. A thermordynamic analysis J. Pharm. Sci., 2009, 99, 1254 – 1264. 20. Murdande, S. B.; Pikal, M. J.; Shanker, R. M.; Bogner, R. H. Solubility advantage of amorphous pharmaceuticals II. Application of quantitative thermodynamic relationships for prediction of solubility enhancement in structurally diverse insoluble pharmaceuticals. Pharm. Res. 2010, 27, 2704 – 2714.


Page 39 of 42


39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

21. Hoffman, J. D. Thermodynamic driving force in nucleation and growth processes. J. Chem. Phys., 1958, 29, 1192 – 1193. 22. Marsac, P. J.; Konno, H.; Taylor, L. S. A comparison of the physical stability of amorphous felodipine and nifedipine systems. Pharm. Res., 2006, 23, 2306-2316. 23. Baird, J. A.; Van Eerdenbrugh, B.; Taylor, L. S. A classification system to assess the crystallization tendency of organic molecules from undercooled melts. J. Pharm. Sci., 2010, 99, 3787 – 3806. 24. Van Eerdenburgh, B.; Baird, J. A.; Taylor, L. S. Crystallization tendency of active pharmaceutical ingredients following rapid solvent-evaporation – classification and comparison with crystallization tendency from undercooled melts. J. Pharm. Sci., 2010, 99, 3826 – 3838. 25. Strickley, R. G. Currently marketed oral lipid-based dosage forms: Drug products and excipients. In Oral Lipid-Based Formulations: Enhancing the Bioavailability of Poorly Water Soluble Drugs, Edited by Hauss, D., CRC Press, 2007. 26. Ananthapadmanabhan, K. P.; Goddard, E. D.; Turro, N. J.; Kuo, P. L. Fluorescence probes for critical micelle concentration. Langmuir, 1985, 1, 352 – 355. 27. Zhao, C-L.; Winnik, M. A. Fluorescence probe techniques used to study micelle formation in watersoluble block copolymers. Langmuir, 1990, 6, 514 – 516. 28. Pozharskaya, G. I.; Kasapova, N. L.; Skripov, V. P.; Kolpakov, Y. D. The spinodal approximation by the method of scattering in (n-hexane + n-tetradecafluorohexane). J. Chem. Thermodynamics, 1984, 16, 267 – 272. 29. Kuldipkumar, A.; Kwon, G. S.; Zhang, G. G. Z. Determining the growth mechanism of tolazamide by induction time measurement. Cryst. Growth Des., 2007, 7, 234 – 242. 30. Bonnett, P. E.; Carpenter, K. J.; Dawson, S.; Davey, R. J. Solution crystallization via a submerged liquid-liquid phase boundary: oiling out. Chem. Commun., 2003, 698 – 699. 31. Bauer, J.; Spanton, S.; Henry, R.; Quick, J.; Dziki, W.; Porter, W.; Morris, J. Ritonavir: An extraordinary Example of Conformation Polymorphism. Pharm. Res., 2001, 18, 859 – 866. ACS Paragon Plus Environment


Page 40 of 42

40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

32. Law, D.; Krill, S. L.; Schmitt, E. A.; Fort, J. J.; Qiu, Y.; Wang, W.; Porter, W. R. Physicochemical considerations in the preparation of amorphous ritonavir-poly(ethylene glycol) 8000 solid dispersions. J. Pharm. Sci., 2001, 90, 1015 – 1025. 33. Ilevbare, G. A.; Liu, H.; Edgar, K. J.; Taylor, L. S. Understanding polymer properties important for crystal growth inhibition – Impact of chemically diverse polymers on solution crystal growth of ritonavir. Crystal Growth and Design, 2012, 12, 3133 – 3143. 34. Andronis, V.; Yoshioka, M.; Zografi, G. Effects of sorbed water on the crystallization of indomethacin from the amorphous state. J. Pharm. Sci., 1997, 86, 346 – 351. 35. Rabel, S. R.; Maurin, M. B.; Rowe, S. M.; Hussain, M. Determination of the pKa and pH-solubility behavior of an ionizable cycle carbamate, (S)-6-Chloro-4-(cyclopropylethynyl)-1,4-dihydro-4(trifluoromethyl)-2H-3,1-benzoxain-2-one (DMP 266). Pharm. Dev. Technol., 1996, 1, 91 – 95. 36. Ramulu, G. Kumar, Y. R.; Vyas, K.; Suryanarayana, M. V.; Mukkanti, K. A new validated liquid chromatographic method for the determination of loratadine and its impurities. Sci. Pharm., 2011, 79, 277 – 291. 37. Shoichet, B. K. Interpreting Steep Dose-Response Curves in Early Inhibitor Discovery. J. Med. Chem.,

2006, 49, 7274 – 7277. 38. Amis, E. J.; Hu, N.; Seery, T. A. P.; Hogen-Esch, T. E.; Yassini, M.; Hwang, F.; Hydrophilic polymers. Associating polymers containing fluorocarbon hydrophobic units. Advances in Chemistry, Vol. 248,

1996, 279 – 302. 39. Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes; John Wiley: New York, 1973. 40. Chen, L-J.; Lin, S-Y.; Huang, C-C.; Chen, E-M. Temperature dependence of critical micelle concentration of polyoxyethylenated non-ionic surfactants. Colloid. Surface. A, 1998, 135, 175 – 181. 41. Schick, M. J. Effect of temperature on the critical micelle concentration of nonionic detergents. Thermodynamics of micelle formation. J. Phys. Chem., 1963, 67, 1796 – 1799. ACS Paragon Plus Environment

Page 41 of 42


41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

42. Flockhart, B. D. The effect of temperature on the critical micelle concentration of some paraffin-chain salts. J. Colloid Sci., 1961, 16, 484 – 492. 43. Stead, J. A.; Taylor, H. Some solution properties of certain surface active N-alkylpyridinium Halides I. Effect of temperature on the critical micelle concentrations. J. Colloid Interf. Sci., 1969, 30, 482 – 488. 44. Kang, K-H.; Kim, H-U.; Lim, K-H.

Effect of temperature on critical micelle concentration and

thermodynamic potentials of micellization of anionic ammonium dodecyl sulfate and cationic octadecyl trimethyl ammonium chloride. Colloid. Surface. A, 2001, 189, 113 – 121. 45. Yalkowsky, S. H. Solubility and solubilization in aqueous media. New York: Oxford University Press,

1999. 46. Sandler, S. I. Chemical and engineering thermodynamics. 3rd Edition, John Wiley and Sons, 1999. 47. Parks, G. S.; Huffman, H.M.; Cattoir, F. R. Studies on glass II. The transition between the glassy and liquid states in the case of glucose. J. Phys. Chem., 1928, 31, 1366 –1379. 48. Parks, G. S.; Snyder, L. J.; Cattoir, F. R. Studies on glass. XI. Some thermodynamic relations of glassy and alpha-crystalline glucose. J. Chem. Phys., 1934, 2:595 – 598. 49. Hancock, B. C.; Parks, M. What is the true solubility advantage for amorphous pharmaceuticals? Pharm. Res., 2000, 17, 397 – 404.



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FOR TABLE OF CONTENTS USE ONLY

Liquid-Liquid Phase Separation in Highly Supersaturated Aqueous Solutions of Poorly-Water Soluble Drugs – Implications for Solubility Enhancing Formulations Grace A. Ilevbare, Lynne S. Taylor

The phase behavior of eight pharmaceutically relevant, poorly-water compounds were investigated and found to undergo LLPS in highly supersaturated solutions. The concentration at which LLPS was observed was comparable to the theoretical amorphous “solubility” of the various compounds. The phase diagram for the ritonavir-water system indicated that LLPS occurs in a metastable region of the phase diagram and is thus a precursor to crystallization.


LiquidâLiquid Phase Separation in Highly Supersaturated Aqueous

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