Assessing Physical Stability of Colloidal Dispersions Using a

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Assessing Physical Stability of Colloidal Dispersions Using Turbiscan Optical Analyzer Yan Sun, Alexandru Deac, and Geoff G.Z. Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01194 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019

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Molecular Pharmaceutics

Assessing Physical Stability of Colloidal Dispersions Using Turbiscan Optical Analyzer Yan Sun1, Alexandru Deac2,3, Geoff G. Z. Zhang2* 1Science

and Technology, Operations, AbbVie, Inc., 1 N. Waukegan Road, North Chicago, IL

60064, United States 2Drug

Product Development, Research and Development, AbbVie, Inc., 1 N. Waukegan Road,

North Chicago, IL 60064, United States 3Current

Address: Department of Industrial and Physical Pharmacy, College of Pharmacy,

Purdue University, West Lafayette, IN 47907, United States

Correspondence: Geoff G. Z. Zhang, email: [email protected], telephone: 1-847937-4702, facsimile: 1-847-937-7756, Drug Product Development, Research & Development, AbbVie, Inc., 1 N. Waukegan Road, North Chicago, IL 60064, United States

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ABSTRACT The physical stability of aqueous colloidal dispersions containing highly concentrated droplets of poorly water-soluble drugs has recently been identified as one of the main considerations in developing amorphous solid dispersions (ASDs). Turbiscan, an instrument based on multiple light scattering technology, was employed for the first time to assess colloidal dispersions with ritonavir as the model compound. The physical instability of ritonavir-rich droplets was monitored directly with and without the presence of candidate polymer additives at different drug concentrations and temperatures. The mechanism of the observed instability was confirmed to be coalescence of liquid droplets, based on the low glass transition temperature of water-saturated amorphous ritonavir determined using a newly developed experimental procedure. Temperature and solvent composition, within the range studied, have little influence on the kinetics of ritonavir coalescence. On the contrary, higher concentration of drug, i.e., more droplets per unit volume, greatly accelerates the coalescence process. In addition, polymers with varying degrees of hydrophobicity resulted in different levels of effectiveness in stabilization which is likely related to the strength of drug-polymer interactions and the corresponding differences in surface adsorption. This work demonstrates that the Turbiscan optical analyzer can be used as a rapid screening tool that provides a first-pass, high-throughput feasibility ranking of different excipients and additives to support the development of ASD formulations.

Keywords: physical stability, coalescence, colloidal dispersions, nanodroplets, stabilization, amorphous, ritonavir, glass transition temperature, Turbiscan


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INTRODUCTION Formulating an oral drug product often requires strategic approaches to overcome challenges in solubility and permeability. Amorphous solid dispersions (ASDs) have become an attractive formulation platform for poorly water-soluble compounds where the advantages include higher drug solubility and faster dissolution rates compared to the crystalline form [1, 2, 3]. Recently, it has been demonstrated that certain ASDs can form colloidal species upon dissolution [4]. The in situ formation of drug-rich droplets is achieved through a process termed liquid-liquid phase separation (LLPS), i.e., spinodal decomposition of immiscible liquids, that spontaneously occurs when the amorphous solubility of the drug is exceeded [5]. This phenomenon constitutes an important aspect in the formulation design of ASDs. Furthermore, there has been an increased interest in understanding the role of these drug-rich droplets in enhancing oral absorption as well as a need to control their physical properties. One such role is their ability to act as a drug reservoir where the free drug concentration in gastrointestinal fluid can be maintained close to or at its maximum during absorption, thereby enabling a maximum and constant flux across the intestinal wall [6]. This effect is due to the combination of higher apparent solubility of amorphous phases and their high specific surface area which translates into fast dissolution rates which, in turn, translates into a higher maximum flux compared to the crystalline form. In order to achieve such reservoir effect, the physical stability of these droplets is critical in maintaining a large specific surface area over the absorption-relevant timeframe. While crystallization has always been a known risk in general, an additional risk associated with these droplets is their tendency to undergo coalescence or agglomeration, which can compromise their effective specific surface area and fast dissolution rates, thus significantly limit drug delivery efficiency. Optimal performance for ASD formulations that exploit these amorphous drug-rich droplets can be achieved through the addition of selective pharmaceutical excipients (polymers and surfactants) that can inhibit crystallization and adequately stabilize these nano-suspensions throughout the absorption process. Using ritonavir as the model compound, Ilevbare et al. confirmed that it is possible to generate physically stable colloidal droplets, from the dissolution of ASDs, which are on the order of 250 to 350 nm in the presence of certain polymers and surfactants [7]. A variety of analytical techniques including UV/vis spectroscopy, dynamic light scattering (DLS), fluorescence spectroscopy, differential scanning calorimetry (DSC), and scanning electron 3 ACS Paragon Plus Environment


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microscopy (SEM) were employed to study the system. The successful utilization of the first two techniques demonstrates that light is an effective way to monitor the size and evolution of these colloidal droplets. To meet the increasing need for rapid development of ASD formulations, the ability to analyze a colloidal dispersion over time with minimal adjustment in the test setup is desirable. To this end, the Turbiscan (Formulaction, L'Union, France) optical analyzer is employed. This instrument utilizes multiple light scattering technology and is becoming increasingly popular for assessing a variety of organic and inorganic systems for the development of products in the food, pharmaceutical, personal care, paint, and oil industries [8] [9]. As has been demonstrated in previous studies in a diverse arena, the main advantage of this technology is its ability to analyze samples ranging from the very dilute to the very concentrated, therefore avoiding the need to adjust the concentration for the sole purpose of performing a measurement. Not only does the elimination of a dilution step save time and effort, it also permits sensitive concentration-dependent studies to be carried out in situ over concentration ranges inaccessible with other techniques. In addition, the Turbiscan Tower version of the instrument can assess six samples simultaneously with minimal manipulation needed. Turbiscan utilizes a near-infrared light source (wavelength = 880 nm) to examine a sample vertically. Two fixed detectors capture the transmission and backscattering signals as a function of position (height from bottom of the vial) and time. These signals are dependent on the particle size and concentration based on Mie theory [9]. The output is a series of intensity profiles that elucidate both the uniformity and evolution of the sample, readily providing indications of particle migration (sedimentation and creaming) as well as particle size changes (coalescence and flocculation) especially at an early stage when the phenomena may not be detectable with other techniques and the naked eye. In this study, colloidal dispersions of ritonavir were prepared using the solvent shift method, where a concentrated solution of ritonavir in organic solvent was added to an aqueous solution, in the presence and absence of polymer. This rapid change of solvent environment creates a supersaturated solution and leads to the formation of ritonavir-rich droplets. Droplets generated with this method have been shown by Ilevbare et al. to be characteristically similar to those produced from the dissolution of a ritonavir-based ASD [7]. The turbidity of the colloidal dispersions was monitored over the course of an hour by using Turbiscan’s mean light transmission signal through the sample. The turbidity of the sample corresponds to the particle 4 ACS Paragon Plus Environment

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size per the Mie theory [9]. The physical stability of ritonavir droplets was assessed as a function of ritonavir concentration, temperature, solvent composition, and polymer additives of varying hydrophobicity. The goal was to demonstrate the feasibility of the Turbiscan multiple light scattering technology as a high-throughput screening tool in aiding the development of ASD formulations in the pharmaceutical industry. MATERIALS Ritonavir, an HIV protease inhibitor, was selected as the model compound (Figure 1) and obtained from AbbVie, Inc. (North Chicago, IL) as a crystal form II powder. Ethanol and methanol were used as organic solvents, and pH 6.8 50 mM phosphate buffer with 0.155 M ionic strength adjustment using NaCl was used as aqueous solvent, and all were obtained from SigmaAldrich (St. Louis, MO). Polymers included polyacrylic Acid (PAA) with an average molecular weight of 1800 g/mol obtained from Sigma-Aldrich (St. Louis, MO). Polyethylene Glycol (PEG) 8000 was obtained from Avantor’s J.T. Baker brand (Center Valley, PA). Hydroxypropyl Methylcellulose Acetate Succinate (HPMCAS) LF, Hydroxypropyl Methylcellulose Acetate Succinate (HPMCAS) MF, Hydroxypropyl Methylcellulose Acetate Succinate (HPMCAS) HF, Polyvinylpyrrolidone (PVP) K29/32 (Plasdone K29/32), and Vinylpyrrolidone-Vinyl Acetate Copolymer (PVPVA) K28 (Plasdone S-630) were obtained from Ashland (Covington, KY). Hydroxypropyl Methylcellulose (HPMC) E5 LV (Methocel E5 Premium LV) and Hydroxypropyl Methylcellulose (HPMC) K100 (Methocel K100 Premium) were obtained from Dow (Midland, MI).

Figure 1. Molecular structure of ritonavir (C37H48N6O5S2, 720.946 g/mol).



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METHODS Ritonavir Amorphous Solubility A µDiss Profiler model R2D with heating block model MB8 (PION, Billerica, MA) and a water bath model 2050-1 (Caron, Marietta, OH) were used to monitor the extinction wavelength of 300 nm for ritonavir as a function of concentration. Amorphous solubility was defined as the concentration at which scattering was detected. Vials containing pH 6.8 buffer were brought to temperature using the heating block while stirring at 300 RPM using cross magnetic stir bars. Parafilm was used to prevent evaporation during temperature equilibration. Ritonavir was dissolved in ethanol to yield a range of standard solutions of varying concentrations. Aliquots of each standard solution were injected in the vials containing pH 6.8 buffer by using a glass gastight syringe with needle (Hamilton, Reno, NV). The final solution volume was kept at 20 mL and the ethanol concentration was kept to either 1% v/v or 5% v/v. A UV probe fitted with a 20 mm path length mirror was inserted in each solution and spectra were collected. Ritonavir Wet Glass Transition Temperature The wet glass transition temperature (Tg) of ritonavir is defined as the Tg of amorphous ritonavir that is saturated with water. Amorphous ritonavir was made by adding approximately 5 g of crystalline ritonavir followed by 500 mL of methanol in a 1 L round bottom flask. The walls of the flask were rinsed with methanol and the solution was sonicated until the ritonavir was completely dissolved. Afterwards, methanol was removed using a rotary evaporator from Buchi (New Castle, DE) equipped with a R-215 rotavapor, V-855 vacuum controller, B-491 heating bath, and V-710 vacuum pump. The evaporation was performed at 55 °C and 50 RPM, where pressure was manually decreased from ambient to full vacuum. Right before full vacuum, the speed was increased to 130 RPM. Ritonavir was left to dry at full vacuum for about 15 minutes before it was moved to a vacuum oven to dry overnight at 40 °C and 0.9 inHg with an external dry ice cold trap. The amorphous ritonavir was then scraped from the flask and ground using a mortar and pestle. The starting moisture content in the amorphous ritonavir was quantified by measuring the loss of weight after drying using a thermogravimetric analysis instrument, Q5000, from TA Instruments (New Castle, DE). Three replicate measurements were performed. The drying 6 ACS Paragon Plus Environment

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procedure was performed in three steps: Isothermal hold for 5 minutes, ramp to 120 °C at 10 °C/min, and another isothermal hold for 30 minutes. To create a range of final water contents, amorphous ritonavir was weighed in several Tzero pans to which various amounts of ultrapure, type I water were added to the pans using a 10 ul gastight, 1800 series, glass syringe from Hamilton Company (Reno, Nevada). The pans were then sealed with Tzero hermetic lids and weighed to obtain the total water. The pans were then placed in an oven at 37 °C. The Tg of the samples was measured by differential scanning calorimetry (DSC) using a Q2000 instrument from TA Instruments (New Castle, DE). The accuracy of the Q2000 was verified by running a 10 °C/min heating ramp on a sample of Indium. The Q2000 cell was preequilibrated to 37 °C prior to removing the amorphous ritonavir sample from the oven. Each sample was transferred from the oven to the Q2000 chamber immediately. After which, the sample was cooled to -15 °C at 25 °C/min, and then was heated to 70°C at 10 °C/min. In a separate study, 2 °C/min and 27.5 °C/min cooling rates were used on a water saturated, amorphous ritonavir sample to evaluate the impact of cooling rate on the measured Tg. No impact was observed, indicating that the composition of the amorphous ritonavir is not changing during the cooling process. Therefore, a 25 °C/min cooling rate was appropriate. During analysis, the end of the enthalpic event was defined as the Tg. Samples with excess water content (~6.5-7.5% w/w) were used to establish the holding time needed to reach equilibrium between the amorphous ritonavir and the excess water. Preparation of Ritonavir Colloidal Dispersions Using Solvent Shift Ritonavir was dissolved in ethanol to yield stock solutions of various concentrations. Polymer solutions were prepared by first equilibrating the polymer powder to the room environment by spreading it on a large weighing boat for a minimum of 11 days. The powder was then manually tumbled in a vial. The amount of moisture was quantified by measuring the loss of weight after drying using a thermogravimetric analysis instrument, Q5000, from TA Instruments (New Castle, DE). The drying procedure was performed in three steps: Isothermal hold for 5 minutes, ramp to 120 °C at 10 °C/min, and another isothermal hold for 30 minutes. Polymer solutions of 100 µg/mL (or ppm) in pH 6.8 buffer were made based on dry weight. Solutions of 30 mL pH 6.8 buffer with and without polymer were equilibrated to temperature in a 50 mL jacketed beaker using an Isotemp 3016 water bath from Thermo Fischer Scientific (Hampton, NH), a Lindberg 7 ACS Paragon Plus Environment


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Blue M magnetic stirrer from Thermo Fischer Scientific, and cross magnetic stirrers. Parafilm was used to prevent evaporation during temperature equilibration. Solvent shift was performed by injecting the ritonavir ethanol solutions into the 30 mL buffer solutions using glass gastight syringes and needles (Hamilton, Reno, NV) mounted onto a 33DDS syringe pump from Harvard Apparatus (Cambridge, MA). The injection rate was such that the ritonavir solution was completely injected in 15 seconds and the stirring speed was sufficient to form a strong vortex. The final set of ritonavir colloidal dispersions consisted of two ritonavir concentrations (70 µg/mL and 100 µg/mL) at 1% ethanol content and another two ritonavir concentrations (83 µg/mL and 113 µg/mL) at 5% ethanol content. Physical Stability of Ritonavir Colloidal Dispersions Using Turbiscan Physical stability measurements of the ritonavir droplets were conducted using the Turbiscan Tower (Formulaction, L'Union, France) optical analyzer. Upon completing the solvent shift procedure, the ritonavir colloidal dispersion was immediately pipetted into a custom glass vial designed for the Turbiscan and loaded into the instrument. Transmission and backscattering intensity profiles as a function of position (height from bottom of the vial) were collected as a function of time in 1-minute intervals for an hour. Due to the very rapid formation and subsequent physical evolution of the droplets, the time between initiating the solvent shift and starting the first scan in the Turbiscan instrument had to be kept consistent to ensure repeatability in the measurement. The time was tracked using a timer and amounted to around 60 seconds for completing this task. Average transmission values were obtained across the height of the samples and plotted as a function of time. RESULTS AND DISCUSSION Physicochemical Properties of Ritonavir Being a Class IV drug per the Biopharmaceutics Classification System (BCS), ritonavir is a large lipophilic molecule that is known to be practically insoluble at pH 6.8 (0.001 mg/mL) and exhibits low absorption limited by its low dissolution rate [10]. In order to design appropriate experiments and analyze the data meaningfully, solubility values of both the crystalline and amorphous phases are necessary for preparing colloidal dispersions of ritonavir. The crystalline 8 ACS Paragon Plus Environment

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solubility of ritonavir in ethanol/water mixtures follows a non-monotonic trend with increasing water content: For form II at 5 °C, the solubility is 19 mg/mL at 99/1 ethanol/water ratio, increases to 61 mg/mL at 85/15, and decreases back to 30 mg/mL at 75/25 [11]. Ritonavir has a dry glass transition temperature (Tg) of around 50 °C and is known to exhibit excellent physical stability [12]. The tendency for the amorphous form to crystallize is low even at temperatures above the Tg. The amorphous solubility of ritonavir, which is known to coincide with the LLPS concentration, was measured in order to perform experiments with known amounts of drug present as colloidal species. The amorphous solubility values were determined at both 25 °C and 37 °C where the physical stability studies were carried out. The solvent environment for the measurements consisted of pH 6.8 aqueous buffer with a small amount of ethanol (1% and 5%) present from carrying out the solvent shift procedure. The data are summarized in Table 1. As expected, the amorphous solubility of ritonavir is higher with higher ethanol content. The results also show that the amorphous solubility at 25 °C and 37 °C with 1% ethanol content are comparable. Hence, given the same total concentration of ritonavir and solvent composition, a similar amount of ritonavir is expected to be present in the dispersed phase at both 25 °C and 37 °C. Table 1.

Amorphous solubility of ritonavir at different temperatures and solvent compositions.

Temperature (°C)

Aqueous Content (% v/v)

Ethanol Content (% v/v)

Ritonavir Amorphous Solubility (µg/mL)

25 37 25

99 99 95

1 1 5

33.77 ± 0.62 32.35 ± 0.52 46.38 ± 0.30

Preparation of the ritonavir colloidal dispersions in this study accounted for the amorphous solubility of ritonavir of around 33 µg/mL and 46 µg/mL for 1% and 5% ethanol content, respectively, so that the final ritonavir droplet concentrations are roughly similar at the two ethanol contents as shown in Table 2.



Table 2.

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Estimated ritonavir droplet concentrations for different solvent compositions.* 1% Ethanol

Total Concentration (µg/mL)

5% Ethanol

Droplet Concentration (µg/mL)

Total Concentration (µg/mL)

Droplet Concentration (µg/mL)

70 ~ 37 83 ~ 37 100 ~ 67 113 ~ 67 *Ritonavir droplet concentration estimations were based on mass balance, i.e., the total concentrations minus the corresponding amorphous solubility values (Table 1) in those solvent media.

Physical Instability of Ritonavir Droplets Instability indicated by Turbiscan results To the naked eye, ritonavir colloidal dispersions first appear to be translucent upon preparation via the solvent shift procedure but quickly turn cloudy. An initial optical scan using the Turbiscan instrument confirms this phenomenon. Figure 2 shows an example of the typical transmission and backscattering profiles obtained from a ritonavir colloidal dispersion. The horizontal axis corresponds to the height of the sample from bottom to top. The collection of profiles is shown over the course of an hour, with blue representing the earliest time point and red the latest time point. Both transmission and backscattering signals, which appear to be uniform across the height, begin at maximum intensities and rapidly decrease to and stabilize at a constant minimum value. This observation clearly points to the physical instability of ritonavir droplets. It is speculated that the potential mechanism of instability is likely coalescence as opposed to agglomeration based on previous knowledge [7] and is to be further justified based on the glass transition temperature of the hydrated ritonavir in this work.

(a)


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(b) Figure 2. Examples of Turbiscan profiles showing changes in the a) transmission and b) backscattering over an hour. Complementary characterization of droplet size by DLS To complement the observations made with Turbiscan, the phenomenon was evaluated quantitatively using DLS with a Zetasizer Nano ZS (Malvern, Worcestershire, UK). Particle size distributions were collected at 25 °C. It can be seen in Figure 3 that there is a fast, approximately linear growth in the average particle size of the ritonavir droplets from around 400 nm to over 2 microns over the course of an hour following their formation. This observation clearly demonstrates the strong tendency for these droplets to undergo the destabilization mechanism of coalescence in an aqueous environment. The ritonavir remained amorphous during this short timeframe and crystallization started many hours after preparation. Once crystallization occurred, large clusters of crystals emerged on the side and/or at the bottom of the vial, and the system became visually clear.



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Figure 3. Physical instability of ritonavir droplets at 100 g/mL and 25 °C. Shown are (a) particle size distributions at several time points and (b) the average particle size as a function of time. Kinetics and mechanism of destabilization and the impact of experimental parameters In order to capture the fast kinetics of coalescence of the ritonavir droplets, Turbiscan was again employed and programmed to monitor the mean transmission signal across the height of the sample continuously over the duration of an hour. Time zero was established as the time at the start of the solvent shift. The collection of the first data point in Turbiscan was consistently initiated at 60 seconds to allow time for loading of the sample into the instrument. To assess the repeatability of this procedure, three replicates were prepared and measured. To evaluate the effect of ritonavir concentration, dispersions containing 70 µg/mL and 100 µg/mL ritonavir with 1% ethanol content at 25 °C were compared. To evaluate the effect of temperature, dispersions containing 100 µg/mL ritonavir with 1% ethanol content at 25 °C and 37 °C were compared. Figure 4 shows the mean transmission values as a function of time for these samples along with the standard deviations (error bars) of the data points. A rapid decrease in the transmission reflects the dispersion turning cloudy and the droplets growing in size. The small error bars demonstrate good repeatability which can be attributed to the consistent sample preparation, including injection speed and volume control of the ritonavir stock solution into the aqueous buffer, the consistent timekeeping (namely the initiation of the Turbiscan measurement at the 60second mark), as well as the consistency of the process itself. It can be observed that the mean transmission curve for the 100 µg/mL ritonavir concentration is lower than that for the 70 µg/mL concentration, which is expected since a greater number of droplets are present in the first case leading to a more turbid dispersion. In terms of temperature, the mean transmission curves at 25 °C and 37 °C are relatively similar in magnitude, suggesting that the increase in thermal motion from the higher temperature is negligible, as expected. In principle, higher thermal motion will increase the collision rate between droplets, which will increase the coalescing rate. The speed of a particle is proportional to the square root of its temperature in Kelvin scale. Assuming other parameters are equal, the increase in speed between the particles at the two temperatures would only be approximately 2%. In addition, the viscosity of the solution media is not expected to be significantly different at these two temperatures. 12 ACS Paragon Plus Environment

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Figure 4. Mean transmission as a function of time for ritonavir droplet dispersions at different ritonavir concentrations and temperature. Next, the impact of solvent composition on the amount of droplets and their coalescence was evaluated. Dispersions utilizing 1% and 5% ethanol content for carrying out the solvent shift were prepared. With knowledge of the amorphous solubility of ritonavir at these two ethanol contents, the total ritonavir concentrations used were adjusted such that the resulting droplet concentrations were approximately the same. Specifically, 70 µg/mL ritonavir with 1% ethanol and 83 µg/mL ritonavir with 5% ethanol were estimated to produce a droplet concentration of around 37 µg/mL based on the amorphous solubility at 1% and 5% ethanol. Similarly, 100 µg/mL ritonavir with 1% ethanol and 113 µg/mL ritonavir with 5% ethanol were estimated to produce a droplet concentration of around 67 µg/mL. The mean transmission curves in Figure 5 clearly show that the dispersions with 1% and 5% ethanol are similar in magnitude. The rate of coalescence also appears to be similar and independent of the solvent composition. Ethanol might affect the thermal motion of the particles by changing the hydrodynamic radius and viscosity. In addition, ethanol itself has a lower thermal motion than water because of its larger size, therefore could reduce the rate of collisions between solvent and particle. However, the result suggests that displacing 4% v/v of water molecules with ethanol molecules is insufficient to alter the thermal motion of the particles significantly.



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Figure 5. Mean transmission signal as a function of time for ritonavir droplet dispersions at different ritonavir concentrations and solvent compositions. The mean transmission curves from Turbiscan show an exponential-like decay over time, which is associated with the kinetics of the ritonavir droplet coalescence. As such, a simple twostep process for the coalescence may be hypothesized to analyze the data [13]. The first step consists of individual droplets (“singlet”) colliding with one another. The second step consists of the collided droplets undergoing coalescence to become a larger droplet (“doublet”). This concept is illustrated in Figure 6. While separation of droplets upon collision can occur, it is assumed that the liquid-like droplets preferentially stick together to complete the coalescence process. As such, only the kinetic time constants associated with the collision and coalescence steps appear in this model. Furthermore, while it is also likely that a fraction of droplets that undergo coalescence will go through further coalescence events to form even larger droplets, the probability and number of droplets that do so can be expected to decrease exponentially with decreasing collision frequency. Since light scattering provided by the Turbiscan instrument can be considered as a mean-field measurement, it is reasonable to approximate the overall behavior without considering beyond a single collision and coalescence event. This model can be considered reasonable for the case where there are no polymer additives and no additional factors influencing the kinetics of the coalescence. In the case where there are drug-polymer interactions that keep the droplets from readily coalescing, a more complicated model is required to interpret the data. The curve fits in Figure 6 provide the trend in the transmission between time zero and 14 ACS Paragon Plus Environment

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the first data point at 60 seconds which is not accessible experimentally. Based on the fits, the time zero transmission values are estimated to be around 68% for the 70 µg/mL ritonavir concentration and 45% for the 100 µg/mL concentration samples. Considering that the pure pH 6.8 aqueous buffer without any ritonavir droplets has a mean transmission value of around 89%, this analysis demonstrates that the formation of the submicron-sized ritonavir droplets is nearly instantaneous upon crossing the amorphous solubility during solvent shift. The fitting parameters to the model 𝐴 + 𝐵(𝑒 ―𝐶𝑡 + 𝑒 ―𝐷𝑡) are listed in Table 3. A simple interpretation of the four parameters A, B, C, and D shows that the value for A + 2B corresponds to the transmission at time zero, while 1/C and 1/D are two independent time constants that describe the characteristic times for the collision and coalescence events. It can be seen that the processes in the 100 µg/mL concentration samples are much faster (with the larger C and D values) than those in the 70 µg/mL samples. This result is expected since a greater number of droplets leads to more collisions and faster coalescence.

Figure 6. Mean transmission signal as a function of time for ritonavir droplet dispersions and corresponding fits to a two-step coalescence kinetic model (All fittings have R2 equal or greater than 0.999). Table 3.

Fitting Parameters to a Two-Step Coalescence Kinetic Model for Ritonavir Droplet Dispersions Condition

A (%)

B (%)

C (sec-1)

D (sec-1)

70 ppm RTV, 25 °C, 1% EtOH

20.3967

23.8961

0.0054

0.0009


6.7253

19.4970

0.0095

0.0017




6.1107

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17.7943

0.0112

0.0021

Understanding the Physical State of Ritonavir Colloidal Species Further justification for the instability mechanism of coalescence that occurs with the ritonavir droplets can be made based on whether the droplets exist in a supercooled liquid or glassy state at the experimental temperatures of 25 °C and 37 °C. For coalescence to occur, the droplets must be liquid-like where the wet glass transition temperature (Tg) is below the experimental temperature. The wet Tg represents the Tg of the drug when saturated with water, which could be significantly lower than the typically cited dry Tg due to the plasticizing effects of water. In these colloidal dispersions where LLPS has occurred, the wet Tg determines the temperature threshold above and below which the drug can exhibit very different physical behavior (liquid-like vs. solid-like) in the aqueous medium. The wet Tg was obtained by saturating amorphous ritonavir samples with water and measuring the Tg by DSC. Samples were prepared by adding different amounts of water to amorphous ritonavir, using a syringe, followed by sealing and equilibrating the samples in an oven at 37 °C for a minimum of four days. The DSC thermograms in Figure 7(a) for 0%, 2.1%, and 4.8% water have similar profiles for the glass transition event, suggesting that these samples were equilibrated at their respective water content. Furthermore, Figure 7(a) shows a gradual decrease in the Tg with increase in water content, as is expected from the plasticizing effect. This is better illustrated in Figure 7(b), where the Tg is plotted as a function of added water. At low water contents, the relationship between Tg and water content agrees quite well with the Gordon-Taylor equation. However, above approximately 5.57%, the Tg remains constant. This behavior can be explained as follows: At low water content the amorphous ritonavir can absorb all of the added water. However, at certain water content the amorphous ritonavir becomes saturated with water and is unable to absorb more water. Additional water added above this saturation point is excess water. Therefore two phases exist at the high water content: a drug-rich phase saturated with water and an excess water phase containing dissolved drug (although the drug concentration is very low). The total amount of drug-rich phase will decrease and the total amount of excess water phase will increase as more water is added. However, the compositions of the two phases remain constant. Therefore, the Tg of the sample does not change above the saturation point. In addition, when there is excess water, the water could freeze upon cooling and melt upon heating. The melting of the ice is observed by 16 ACS Paragon Plus Environment

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the sharp endotherm at about 0 °C in Figure 7(a) for the 9.1% w/w sample. A melting point similar to that of pure water is expected since the ritonavir solubility in water is too low to depress the freezing/melting point to the extent that can be experimentally discerned. In summary, the Tg values that are obtained from the samples saturated with water correspond to the wet Tg of amorphous ritonavir, which is determined to be 14.06 °C ± 0.23 °C, ~23 °C and ~11 °C lower than the experimental temperatures of 37 °C and 25 °C, respectively. It is likely that the wet Tg is even lower when residual ethanol is present from the solvent shift process. This result thus confirms that the ritonavir colloidal dispersions in fact consist of liquid droplets as opposed to glassy particles. The liquid-like nature of these droplets also explains their highly spherical geometry as observed by optical microscopy. Most importantly, any destabilization mechanism resulting in growth in size of these droplets can be affirmatively considered as coalescence rather than agglomeration. Stabilization of Ritonavir Droplets Using Polymeric Additives To demonstrate the utility of Turbiscan as a screening tool for formulation of ASDs, the effects of various polymeric additives on the physical stability of the ritonavir colloidal droplets were assessed at different ritonavir concentrations and temperatures. The mean transmission was monitored on colloidal dispersions containing 70 µg/mL and 100 µg/mL ritonavir with 1% ethanol content in the presence of 100 µg/mL polymer in the aqueous medium. Figures 8 to 10 show the transmission curves for the different polymers. As can be seen in general, hydrophilic polymers such as PAA and PEG are ineffective in improving the physical stability of the ritonavir droplets, with the droplets coalescing at a rate comparable to that of the control (no polymers present). On the other hand, hydrophobic polymers such as the HPMCAS HF grade can significantly reduce the tendency for the ritonavir droplets to coalesce, as reflected by a much slower decrease in the transmission curve. Other polymers such as PVP, PVPVA, HPMC, and HPMCAS (LF and MF grades) also provide some level of stabilization, though not as effective as the HPMCAS HF. These results are similar for both the 70 µg/mL and 100 µg/mL ritonavir concentrations that were studied and are consistent with the idea that the hydrophobic ritonavir has a preference to interact with hydrophobic polymers more than hydrophilic polymers. Hydrophobic polymers adsorb more readily to the ritonavir droplet surface, preventing it from coalescing with another droplet [7]. 17 ACS Paragon Plus Environment


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0%

2.1%

4.8%

(a)

9.1%

(b)

Figure 7. DSC results from sealed amorphous ritonavir samples, containing various amounts of water, that were held in an oven at 37 °C for at least four days, where (a) shows an overlay of the obtained thermograms and (b) shows a plot of Tg as a function of water content, where

is the Gordon-Taylor fit and

is a flat line

representing the average of the data points above 5.57% water.


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Figure 8. Mean transmission signal as a function of time for ritonavir droplet dispersions (70 µg/mL ritonavir, 25 °C, 1% EtOH) containing different polymers (100 µg/mL).

Figure 9. Mean transmission signal as a function of time for ritonavir droplet dispersions (100 µg/mL ritonavir, 25 °C, 1% EtOH) containing different polymers (100 µg/mL).



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Figure 10. Mean transmission signal as a function of time for ritonavir droplet dispersions (100 µg/mL, ritonavir, 37 °C, 1% EtOH) containing different polymers (100 µg/mL). The transmission profiles and the rate of coalescence are also similar at both 25 °C and 37 °C for all polymeric additives studied. Such behavior is expected since the amorphous solubility of ritonavir is similar at both temperatures, producing a similar amount of droplets. The only exception can be seen in Figure 11 with the HPMCAS HF grade polymer. Here, the transmission curves begin at a similar level but diverge after around 600 seconds (10 minutes) in the data collection, with the transmission level remaining higher for the 37 °C than the 25 °C measurement. This result indicates that the droplets are better stabilized and remain small at 37 °C than at 25 °C. Since there are a similar amount of ritonavir droplets at both temperatures, the other factor causing the difference could be traced to the poor aqueous solubility of the HPMCAS HF polymer itself. The highly hydrophobic polymer chains of HPMCAS HF are more coiled at 25 °C than at 37 °C, thus at 25 °C a fewer fraction of the polymer chains are able to interact and adsorb to the surfaces of the ritonavir droplets than at 37 °C. As the droplets undergo coalescence, the surface-to-volume ratio decreases. This process reaches a point where at 37 °C there are a sufficient amount of HPMCAS HF polymer to cover a large portion of the droplet surfaces, but at 25 °C there is not enough polymer to do the same. The end effect is that the droplets continue to grow at 25 °C, decreasing the transmission signal, but effectively stop 20 ACS Paragon Plus Environment

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growing at 37 °C, leveling off the transmission signal. The other polymers do not exhibit this difference as their aqueous solubility is greater; therefore do not have such temperature dependence.

Figure 11. Mean transmission signal as a function of time for ritonavir droplet dispersions with and without the prescence of HPMCAS HF at different temperatures. The formation of colloidal droplets of poorly water-soluble compounds, such as ritonavir, by LLPS, has been previously observed and investigated by Ilevbare et al. and others. In order to exploit the unique properties of these droplets in ASD formulations and provide a rapid assessment of their physical stability, a new measurement approach based on the principle of multiple light scattering was applied in this work. By using Turbiscan to monitor the mean light transmission signal through a sample over time, precise changes in the sample turbidity were directly linked to and quantified into the kinetics of physical destabilization. With the attractive perspective that these droplets can enhance oral absorption, a fast and practical tool is indispensable in designing amorphous solid dispersions (ASDs). While characterization tools such as DLS and microscopy are essential for scientific research, a rapid screening approach such as the one provided by Turbiscan must be undertaken during the selection of formulation candidates when time is critical and many options are available. CONCLUSIONS 21 ACS Paragon Plus Environment


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The physical instability of ritonavir colloidal droplets generated via LLPS was evaluated using the Turbiscan optical analyzer, an instrument based on multiple light scattering. By directly monitoring the light transmission signal through the sample over time, the data were used to analyze the kinetics of the growth of the droplets. The mechanism of instability was attributed to coalescence, which was confirmed through determination of the wet glass transition temperature using a newly developed experimental procedure and demonstrating that the droplets are in fact liquid-like at the temperature of the stability assessment. The impact of temperature, ritonavir concentration, solvent composition, and polymer additives on the physical stability of the droplets was assessed. The results indicate that differences in temperature (25 °C vs. 37 °C) and solvent composition (1% vs. 5% ethanol) did not have a significant effect on the coalescence kinetics of the droplets. However, the ritonavir concentration, translating to a specific number of droplets, was observed to have a considerable impact on the kinetics. More droplets resulted in a higher rate of collision and faster coalescence, as indicated by analysis of the transmission curve using a two-step kinetic model. The type of polymer additive used was also observed to dictate the coalescence behavior. Polymers that are more hydrophobic were seen to be more effective in stabilization and keeping the droplet size small, while more hydrophilic polymers were less effective in slowing down or preventing droplet growth. Crystallization of ritonavir was not an issue over these short experimental times, so all changes in the transmission signal was directly attributed to the coalescence phenomenon. This study demonstrates that the Turbiscan multiple light scattering instrument can be successfully adopted as a high-throughput, rapid screening tool for the development of ASD formulations in the pharmaceutical industry. ACKNOWLEDGEMENTS Y.S. would like to acknowledge Drug Product Development at AbbVie for the internal crosstraining opportunity during which this work was carried out. DISCLOSURE


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The design, study conduct and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, writing, reviewing, and approving the publication. Y.S., A.D., and G.G.Z.Z. are employees or former employees of AbbVie and may own AbbVie stock. SUPPORTING INFORMATION More information regarding the experimental procedure and analysis of the differential scanning calorimetry (DSC) thermograms for determining the wet glass transition temperature (Tg) of water-saturated amorphous ritonavir. REFERENCES [1] Zhang, G. G. Z., Zhou, D., “Crystalline and Amorphous Solids”. In: Qiu, Y., Chen, Y., Zhang, G. G. Z., Liu, L., Porter, W. R. (eds), Developing Solid Oral Dosage Forms: Pharmaceutical Theory and Practice, Elsevier, 25-60 (2009). [2] Bellantone, R. A., “Fundamentals of Amorphous Systems: Thermodynamic Aspects”. In: Shah, N., Sandhu, H., Choi, D. S., Chokshi, H., Malick, A. W. (eds), Amorphous Solid Dispersions: Theory and Practice, Springer, 3-34 (2014). [3] Imaizumi, H., Nambu, N., Nagai, T., “Stability and Several Physical Properties of Amorphous and Crystalline Forms of Indomethacin”, Chem. Pharm. Bull., 28, 2565-2569 (1980). [4] Alonzo, D. E., Gao, Y., Zhou, D., Mo, H., Zhang, G. G. Z., Taylor, L. S., “Dissolution and precipitation behavior of amorphous solid dispersions”, J. Pharm. Sci., 100, 3316-3331 (2011). [5] Taylor, L. S., Zhang, G. G. Z., “Physical chemistry of supersaturated solutions and implications for oral absorption”, Advanced Drug Delivery Reviews, 101, 122-142 (2016).



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[6] Indulkar, A. S., Gao, Y., Raina, S. A., Zhang, G. G. Z., Taylor, L. S., “Exploiting the phenomenon of liquid-liquid phase separation for enhanced and sustained membrane transport of a poorly water-soluble drug”, Mol. Pharmaceutics, 13, 2059-2069 (2016). [7] Ilevbare, G. A., Liu, H., Pereira, J., Edgar, K. J., Taylor, L. S., “Influence of additives on the properties of nanodroplets formed in highly supersaturated aqueous solutions of ritonavir”, Mol. Pharmaceutics, 10, 3392-3403 (2013). [8] Mengual, O., Meunier, G., Cayre, I., Puech, K., Snabre, P., “TURBISCAN MA 2000: multiple scattering measurement for concentrated emulsion and suspension instability analysis”, Talanta, 50, 445-456 (1999). [9] Buron, H., Mengual, O., Meunier, G., Cayre, I., Snabre, P., “Optical characterization of concentrated dispersions: applications to laboratory analyses and on-line process monitoring and control”, Polym. Int., 53, 1205-1209 (2004). [10] Zhang, G. G. Z., Law, D., Schmitt, E. A., Qiu, Y., “Phase transformation considerations during process development and manufacture of solid oral dosage forms”, Advanced Drug Delivery Reviews, 56, 371-390 (2004). [11] Bauer, J., Spanton, S., Henry, R., Quick, J., Dziki, W., Porter, W., Morris, J., “Ritonavir: An extraordinary example of conformational polymorphism”, Pharmaceutical Research, 18, 859866 (2001) [12] Zhou, D., Grant, D. J. W., Zhang, G. G. Z., Law, D., Schmitt, E. A., “A calorimetric investigation of thermodynamic and molecular mobility contributions to the physical stability of two pharmaceutical glasses”, Journal of Pharmaceutical Sciences, 96, 71-83 (2007). [13] Saether, O., Sjoblom, J., Dukhin, S. S., “Droplet Flocculation and Coalescence in Dilute Oil-in-Water Emulsions”. In: Friberg, S. E., Larsson, K., Sjoblom, J. (eds), Food Emulsions, Marcel Dekker (2004). 24 ACS Paragon Plus Environment

Assessing Physical Stability of Colloidal Dispersions Using a

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