Self-Association of Rafoxanide in Aqueous Media and Its Application

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Self-Association of Rafoxanide in Aqueous Media and Its Application in Preparing Amorphous Solid Dispersions Fan Meng, Tongzhou Liu, Elizabeth Schneider, Shehab Alzobaidi, Marco Gil, and Feng Zhang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00068 • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

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Molecular Pharmaceutics

Self-Association of Rafoxanide in Aqueous Media and Its Application in Preparing Amorphous Solid Dispersions

Fan Meng1, Tongzhou Liu1, Elizabeth Schneider1, Shehab Alzobaidi2, Marco Gil3 and Feng Zhang1

1

College of Pharmacy, the University of Texas at Austin, 2409 University Ave, Austin, TX 78712, USA

2

Department of Chemical Engineering, the University of Texas at Austin, 200 E Dean Keeton St, Austin,

TX 78712, USA 3

Hovione LLC, 40 Lake Drive, East Windsor, NJ 08520, USA

* Corresponding Author: Feng Zhang College of Pharmacy The University of Texas at Austin 2409 University Avenue, A1920 Austin, TX 78712 Phone: (512) 471-0942 Fax: (512) 471-7474 Email:[email protected]

1 ACS Paragon Plus Environment


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1

ABSTRACT

Our primary objective is to characterize the self-association of rafoxanide in alkaline media. The second objective is to illustrate the feasibility of using rafoxanide micellar solution as the feed solution to prepare amorphous solid dispersion via spray drying. Rafoxanide is a poorly water-soluble drug. It is a weak acid, and its poor aqueous solubility is due to its hydrophobicity. The surface-active property of rafoxanide has not been previously reported. It was discovered that the addition of a small percentage of organic solvents is required to elevate the solubility of rafoxanide above the critical micelle concentration to allow for the formation of micelles. Our fluorescence decay study confirms the self-association of rafoxanide in a cosolvent consisting of 70%, v/v, NaOH solution and 30%, v/v, acetone. The position of each functional group in the micellar structures using the 1H NMR technique was identified. The critical micelle concentration of rafoxanide in the cosolvent is determined to be 302 µg/mL using a surface tension method. The solubility of rafoxanide in 0.1 N NaOH solution is less than 11 µg/mL. Interestingly, the apparent solubility increased to 38,400 µg/mL in the presence of 30% acetone as the result of micelle formation. This unique solubility characteristic makes it feasible to prepare rafoxanide amorphous solid dispersions by spray drying a predominantly aqueous (70% 0.1 N NaOH solution and 30% acetone) based feed solution. Povidone and copovidone were both used as polymeric carriers. Based on solid-state characterization, including differential scanning calorimetry, X-ray powder diffraction, and hot-stage polarized light microscopy, our results indicate that rafoxanide solid dispersions prepared using this novel process are amorphous. Approximately 750-fold increase in the concentration of rafoxanide in aqueous media at pH 6.8 was achieved with the amorphous solid dispersions. KEYWORDS: rafoxanide, self-association, surface active, amorphous solid dispersion, solubilization, spray drying


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2

INTRODUCTION

A number of drug substances are surface active[1]. Surface-active compounds usually possess an ionic or nonionic polar head group and a hydrophobic tail group. In aqueous media, as the concentration reaches a certain level, surface-active drugs self-associate into organized aggregates[2]. A few studies have investigated the effect of self-association on the physicochemical properties of surface-active drugs. Surakitbanharn et al. reported that dimerization of dexverapamil at concentrations above 10-3 M increases dexverapamil solubility and decreases its apparent pKa[3]. Tavano et al. observed that cromolyn sodium self-associates into different types of supramolecular aggregates, and certain types of these aggregates demonstrate better diffusion through a biological membrane[4]. Even though self-association (e.g., micelle formation) can increase apparent solubility, the drug concentration must exceed the critical micelle concentration in order for self-association to occur[5]. Since micellization could be used to achieve high concentration of surface-active drugs, it is of our interest to study the feasibility of preparing spray-dried amorphous solid dispersions of poorly water-soluble, surface-active drugs using its micelle solution. Depending on the flexibility of the hydrophobic groups, the association patterns of surface-active drugs can be divided into two categories. Surface-active molecules that contain rigid hydrophobic groups, such as planar rings or heteroaromatic rings, tend to stack face-to-face in a continuous pattern[6]. In contrast, surface-active molecules that contain flexible hydrophobic groups, such as diphenylmethane, tend to associate as micelles, in which the hydrophobic groups are sequestered from the aqueous medium by a surrounding shell of the hydrophilic head groups[1]. The aggregation number, which is the average number of molecules in a micelle, depends on two factors: (a) the hydrophobicity of the nonpolar tails and (b) the repulsion among the polar head groups. It has been reported that micellization is retarded by the presence of high levels of organic solvent in aqueous media. Organic solvents minimize the repulsion between the polar aqueous media and nonpolar hydrophobic tails, thus reducing the tendency of tails to transfer from the bulk solution into the micellar core[7].



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Rafoxanide (RAF) is the model compound in this study. Figure 1a presents the chemical structure of RAF. It is a derivative of salicylanilides that is mainly used for veterinary parasite control[8]. RAF is a poorly water-soluble, weakly acidic drug. Its solubility in water is less than 0.1 µg/mL and remains less than 11 µg/mL even in a 0.1 N NaOH solution at pH 13. The absorption of RAF is solubility limited. The current commercial RAF product is formulated as an oral suspension. Shoa’a invented a micellar solution containing RAF and a nonionic surfactant, and this formulation demonstrated improved absorption[9]. We hypothesized that RAF is surface active in alkaline media, in which the 2-hyroxy-3,5-diiodobenzene group becomes ionized. The surface-active properties of RAF have never been reported. As discussion earlier, amorphous solid dispersion of RAF might be a suitable feed solution for preparing amorphous solid dispersion to enhance the kinetic solubility of RAF. Amorphous solid dispersions (ASDs) have been widely applied to improve the oral bioavailability of BCS Class II and Class IV compounds by increasing their kinetic solubility[10]. In ASDs, the hydrophobic drug is converted into a high-energy amorphous state and dispersed at the molecular level in polymeric matrices. The polymer inhibits drug crystallization and precipitation through physical interaction (e.g., hydrogen bonds, Van der Waals forces) in the solid state and in aqueous environments, resulting in a sustained and elevated supersaturation[11]. One common technique to prepare ASD is spray drying. In the spray drying process, a feed solution containing the drug and polymer is atomized to fine droplets and sprayed into a hot gas stream. As the solvent evaporates, the atomized droplets convert into a fine powder[12]. The solvents for the feed solution preparation have been limited to volatile organic solvents (e.g., acetone, methanol, ethanol, and dichloromethane). The use of aqueous media to prepare feed solutions for spray-dried ASDs has not been reported. The objectives of this study are: (1) to understand the self-association mechanisms of RAF in solution state, (2) to investigate the impact of RAF self-association on its apparent solubility, and (3) to evaluate the feasibility of preparing amorphous RAF ASDs using its micellar solution.


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The surface tension method, the fluorescence lifetime method, and the 1H NMR technique were applied to study the self-association of RAF in a cosolvent consisting of an alkaline aqueous medium and acetone. The apparent solubility of RAF as a function of cosolvent composition was measured, and the mechanisms for its unique solubility profile were explained. X-ray powder diffraction, differential scanning calorimetry, hot-stage polarized light microscopy, and non-sink dissolution testing were used to characterize the spray-dried RAF ASDs. . 3

Materials and Methods

Rafoxanide was purchased from ShenZhen Nexconn Pharmatechs Ltd. (ShenZhen, China). Kollidon® VA64 and Kollidon® 25 N.F. were kindly donated by BASF Chemical Company (Florham Park, NJ, USA). HPLC grade acetonitrile was purchased from Fisher Scientific (Pittsburgh, PA, USA). All other chemicals used in this study were ACS grade or higher. 3.1 3.1.1

Methods RAF solubility

The equilibrium solubility of RAF was determined at ambient condition (25ºC). Excess amounts of RAF were added to vials that contained 10 mL of various media (e.g. 0.1N NaOH solution, acetone, acetonitrile, methanol, dichloromethane and a mixture of two solvents). After constant mixing using a magnetic stir bar for 48 hours, the content was centrifuged at 1,400 g force using a Microfuge® 18 Centrifuge (Beckman Coulter, Brea, CA, USA) for 10 minutes. The supernatant was either used as is or diluted using acetonitrile prior to HPLC analysis. We determined the X-ray diffraction pattern of the residual solid to detect any change in the crystalline form of the undissolved RAF. 3.1.2

Fluorescence lifetime

The fluorescence study was performed at room temperature using an RF-5301PC fluorimeter (Shimadzu, Durham, NC, USA). Coumarin-102 (C102) was used as the fluorophore. The excitation wavelength (λex) was set at 400 nm, and the emission wavelength (λem) was set at 485 nm. Both the excitation and emission slits were set to 3 nm. We determined the fluorescence lifetime from time-resolved intensity decays using 5 ACS Paragon Plus Environment


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the time-correlated single-photon counting (TCSPC) method with a picosecond diode laser at 402 nm (nanoLED-05A, IBH, UK) as the light source. The decays were analyzed using IBH DAS-6 decay analysis software (HORIBA Scientific, NJ, USA). The goodness-of-fit was evaluated by the χ2 criterion and by a visual inspection of the residuals of the fitted function to the data. 3.1.3

Nuclear magnetic resonance spectroscopy

1D 1H NMR spectra were acquired on a Varian® NMR 600 MHz Spectrometer (Agilent Inc., Palo Alto, CA, USA) at 25 °C using standard sequences. We prepared RAF solutions that contained different concentrations of RAF (i.e., 0.1, 0.2, 0.5, 1, 2, 5, and 10 mg/mL) in deuterated media. Solutions were then transferred to 5 mm NMR tubes for NMR data acquisition. Chemical shifts were referenced to residual solvent, H2O, at 4.61 ppm. RAF proton chemical shift assignments in solution were derived from the calculation of the coupling constant and COSY. 1H NMR spectra were acquired using an Agilent Direct Drive 600 spectrometer, operating at a proton frequency of 599.75 MHz (14.1 T) at 298 K, equipped with an AutoX DB probe. We used the double-stimulated echo pulse program with bipolar gradient pulses and a longitudinal eddy current delay. The relaxation delay was 2 s, the finite pulse decays were collected into 64,000 complex data points, and 16 scans were acquired for each sample. The gradient pulse strength was increased from 5% to 95% of the maximum strength of 60 Gauss cm−1 in 50 steps. The squared gradient pulse strength was linearly distributed. 3.1.4

Surface tension

We measured the surface tension of a concentration series of RAF solutions in 70%, v/v, 0.1 N NaOH solution and 30% acetone at ambient pressure and 25 °C. We used a Theta™ Optical Tensiometer apparatus (Biolin Scientific, Linthicum Heights, MD, USA) for these measurements. Surface tension was obtained using axisymmetric drop-shape analysis of a pendant surfactant solution drop. The droplet was initially generated using a needle and was allowed to equilibrate for 3–10 min with air until a steady-state measurement was obtained. To minimize acetone evaporation from the solution, the solution droplets were contained in a glass vial during measurement. The droplet shape was analyzed by the OneAttension


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software package (Biolin Scientific, Linthicum Heights, MD, USA) using the Young–Laplace equation. A minimum of three measurements for each condition were recorded, and the average value was reported. The surface tension measurement was reproducible within ± 0.5 mN/m. 3.1.5

Differential scanning calorimetry

We used differential scanning calorimetry (Model DSC Q20, TA Instruments, New Castle, DE, USA) to characterize the thermal properties of RAF drug substances and spray-dried ASDs. The instrument was operated under a dry nitrogen purge (50 mL/min) with the RCS40 (TA Instruments, New Castle, DE, USA) refrigerated cooling system accessory. The instrument was calibrated with an indium standard. The sample of approximately 5 mg was weighted into a standard DSC pan and crumped with hermetic lid. The experiment was conducted at a temperature ramp of 10 °C/min from 25–200 °C. TA Universal Analysis 2000™ software was used for data analysis. 3.1.6

Powder X-ray diffractometry

We investigated the X-ray pattern of the powder samples using a Benchtop X-ray diffraction instrument, the Model Miniflex 600 (Rigaku, Woodlands, TX, USA), using primary monochromated radiation (CuK radiation source, λ = 1.54056 Å). The instrument was operated at an accelerating voltage of 40 kV and at 15 mA. We subjected all samples to the same program: They were scanned over a 2θ range of 5–60° at a step size of 0.02°/s, with a dwell time of 2 s and scan speed of 1°/min. 3.1.7

Hot-stage polarized light microscopy

HSPLM studies were conducted using a Leica DMPL polarizing optical microscope (Leica Microsystems Wetzlar Gmbh, Wetzlar, Germany). Samples were placed between a glass slide and a cover glass with one drop of silicon oil. A Linkham THMS600 hot stage (Linkham Scientific Instruments Ltd., Surry, England) was used to heat the samples from 25 °C to 200 °C at 10 °C/min. The morphology change during the heating process was recorded by digital camera for further analysis.



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3.1.8

Spray drying

A cosolvent consisting of 70% 0.1 N NaOH and 30% acetone was used as the solvent to prepare feed solutions. The total solid content of the feed solutions was 4%, w/v. The solutions were spray-dried using a Buchi mini B290 spray dryer coupled with inert loop B-295 (Buchi, Flawil, Switzerland). The feed solution was processed with an inlet temperature of approximately 100 °C to maintain a 70 °C outlet temperature, 100% aspiration (475 L/h) heated nitrogen gas, a 5 mL/min feed rate, and a condenser temperature of 5 °C. Following spray drying, the ASDs underwent secondary drying in a vacuum oven overnight at 30 in. Hg and 40 ºC to remove residual solvent. 3.1.9

Non-sink dissolution testing

We conducted in vitro release testing of RAF spray-dried ASDs with a USP dissolution apparatus II [13](Vankel dissolution apparatus, Model 7000, Palo Alto, CA, USA). The paddle speed was set at 50 rpm. We used 1 L of 0.1 N hydrochloric acid solution at pH 1.2 and 37 °C as the dissolution medium. A sample of 300 mg spray-dried dispersion, containing 75 mg rafoxanide, was introduced into each vessel. We collected 1-milliliter dissolution samples at 5, 15, 30, 45, 60, 90, and 120 min, and we filtered the samples through a 0.45 µm PTFE membrane filter. All dissolution testing was performed in triplicate. RAF samples were analyzed using the HPLC method. 3.1.10 HPLC method for RAF concentration analysis The concentration of RAF was determined using a reversed-phase HPLC method. Hypersil® Gold C18, 5x30 mm, 3 µm (Thermo Scientific, Waltham, MA, USA) was used for separation. An 85%, v/v, water and 15%, v/v, acetonitrile mixture containing 0.05% trifluoroacetic acid was used as the mobile phase. The flow rate was set at 1.0 mL/min, and the injection volume was 10 µL. A UV detector (Waters® 2998 PD detector, Milford, MA, USA) was used to quantitate at 485 nm. The retention time of RAF was 4.5 min. The quantitation limit of the method is 0.1 µg/mL.


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4 4.1

Results and Discussion Critical Physicochemical Attributes of RAF

The chemical structure of RAF and its critical physicochemical properties are presented in Figure 1 and Table 1, respectively. The RAF drug substance is a crystalline material with a melting point of 175.4 °C and a glass transition temperature of 61.6 °C. RAF is a weak acid with a pKa of 4.6. RAF is a poorly water-soluble drug with a LogD of 6.76 in a pH 7.4 buffer[14]. Its solubility is less than 0.1 µg/mL in purified water, and this increases to 10.7 µg/mL in a 0.1 N sodium hydroxide (NaOH) solution. The low solubility of RAF in aqueous media is mainly attributed to its high LogD[15]. The high LogD of RAF is attributed to its three benzene rings as well as its intramolecular hydrogen bonding. Intramolecular hydrogen bonding has been found to decrease the aqueous solubility and increase the lipophilicity of organic compounds[16]. The salicylamide group in RAF can form intramolecular hydrogen bonds by two different mechanisms: (a) between the amide carbonyl oxygen and phenol proton (Figure 1b) and (b) between the phenol oxygen and the amide proton (Figure 1c and Figure 1d). We hypothesized that RAF is surface active, following our solubility studies in mixtures of alkaline aqueous media and organic solvents, and a close examination of its chemical structure. Because RAF has an intrinsic solubility in alkaline aqueous media that is below the critical micelle concentration, the surface-active property of RAF was never realized until the current study. With the addition of a low level of organic solvents, the intrinsic solubility of RAF exceeded the critical micelle concentration required for micellization. As a result, the high apparent solubility of RAF was achieved in a cosolvent that consisted of alkaline aqueous medium and a low percentage of organic solvent. We observed that the solubility of the RAF 0.1 N NaOH solution increased drastically with the addition of organic solvents such as acetone and acetonitrile. The highest apparent solubility of RAF is achieved with an acetone volume fraction of 30%. The solubility of RAF increased 2,850- or 3,589-fold, from 10.7



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µg/mL to 30.5 or 38.4 mg/mL with the addition of 30%, v/v, of acetonitrile or acetone into a 0.1 N NaOH solution, respectively (Table 1). In the current study, it was confirmed using a powder X-ray diffraction technique, that the crystalline form of the RAF solid remained the same throughout the solubility study. Upon further examination of the chemical structure of RAF and a preliminary study using the Ultra Filtration of RAF (Table S1 in the supporting information), we hypothesized the following: (a) RAF is surface active and can self-associate to form micelles in solution state. (b) The intrinsic solubility of RAF in alkaline aqueous media is below the critical micelle concentration, and this low solubility inhibits RAF from aggregating. (c) The increase in the intrinsic solubility of RAF is due to the presence of organic solvents that allow for the formation of micelles, which further enhance RAF solubility. Similar phenomena of solvent effects on micellization have been reported by Fisher et al.[17]. Using cosolvents that consist of aqueous media and organic solvents, they prepared supersaturated micelle solutions of amphiphilic compounds, the aqueous solubility of which were below the critical micelle concentration. RAF solubility behavior in alkaline aqueous medium/acetone cosolvent system would enable us to use a predominantly aqueous medium (i.e., 0.1 N NaOH solution/acetone cosolvent) to prepare amorphous solid dispersions via spray drying. The first section of the current study focuses on studying the self-association of RAF in 0.1 N NaOH solution and acetone cosolvent. The second section discusses the feasibility of using this predominantly aqueous feed solution to prepare RAF ASDs. 4.2 4.2.1

Characterization of RAF micelles in 0.1 N sodium hydroxide–acetone cosolvent system Critical micelle concentration of RAF

The critical micelle concentration (CMC) is the minimum concentration of a surface-active compound in a bulk phase, above which the surface-active compound begin to self-associate to form micellar structures. The CMC of surface-active compounds can be determined using various methods, including surface tension measurement[18], 1H NMR[19], conductometry[20], and fluorescence spectroscopy[21].


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We used a Theta™ optical tensiometer to measure CMC of RAF in a cosolvent consisting of 70%, v/v, 0.1 N NaOH and 30%, v/v, acetone. Figure 2 presents the surface tension of the RAF solution as a function of the logarithm of the RAF concentration. Below the CMC, the surface tension of RAF solutions decrease as the concentration increases, and there is a linear correlation between the surface tension and the logarithm of the RAF concentration. Once RAF concentration exceeds the CMC, the surface tension reaches a plateau. The intersection between the linear regression line of the concentration-dependent region and the straight line that passes through the plateau represents the CMC point. The CMC of RAF was determined to be 302 µg/mL in the current study. This result agrees well with the results from fluorescence decay study (i.e., approximately 200 µg/mL). 4.2.2

Fluorescence decay study

Both steady-state and lifetime fluorescence measurements are commonly used to investigate molecular association in solution state. In steady-state fluorescence measurement, the measured parameters are intensity-weighted averages of the underlying decay processes[22]. Complex systems (e.g., proteins, polymers, surfactants) exhibit multiple structural domains with fluorophores in each domain that exhibit their own characteristic fluorescence decay. When the steady-state fluorescence measurement is used, the information regarding each specific domain is obscured[23]. The fluorescence lifetime measurement, also known as the fluorescence decay method, was developed to obtain domain-specific information. Fluorescence lifetime measurement has been commonly used in areas such as biochemistry[24], polymer science[25], and physical chemistry[26] for more than thirty years, but its application in pharmaceutics has not been widely explored. We applied fluorescence lifetime measurement to investigate the self-association of RAF using Coumarin-102 (C102) as the fluorophore. C102 has been extensively used for monitoring the polarity of its local environment in various solvents[27]. The change in C102 lifetime indicates a change in the polarity of the local environment. 11 ACS Paragon Plus Environment


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The intensity of fluorescence decaying signal at time (t) can be calculated from the multi-exponential model[28]:

= ∑ × − ....................................................................................................... Equation 1

where and are the amplitude and fluorescence lifetime, respectively, of individual exponential components that represent individual domains; M is the total number of exponential components of the fluorescence decay, and is indicative of the fraction of fluorophore present in each domain. Fluorescence decay profiles are presented in Figure 3, and various parameters of fluorescence decay are summarized in Table 2. As shown in Figure 3a, a single-exponential decay that corresponds to a single lifetime was observed across the entire concentration range of the acetone-based RAF solutions. Singleexponential fluorescence decay indicates that the fluorescent probe exists in a single environment[29]. In the cosolvent, the fluorescence decay is single-exponential at a low concentration range (50 µg/mL and 100 µg/mL) but becomes bi-exponential at a high concentration range (200–10,000 µg/mL). Biexponential decay at a high concentration range indicates that C102 existed in two different environments (i.e., the bulk phase and the core of self-associated RAF)[29]. In the bi-exponential decay, a short and a long lifetime corresponds to the lifetime of C102 in the bulk phase and the self-associated RAF domain, respectively. We also conclude from the fluorescence lifetime study that the critical CMC of RAF was around 200 µg/mL. For the two fluorescence lifetime decays detected in RAF solutions in the cosolvent, the amplitude of the shorter lifetime, α2, is indicative of the fraction of the fluorophore in the hydrophobic region of selfassociated RAF. And, the amplitude of the longer lifetime, α1, corresponds to the fluorophore in the bulk solution. The observation that α2 increases with increasing concentrations of RAF implies a higher degree of RAF self-association at higher RAF concentrations. Aggregation of RAF in the cosolvent-based solutions was also confirmed with the ultrafiltration study. The results are summarized in Table S1 of the supporting information.


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4.2.3 1

Location of different functional RAF groups in micelles

H NMR was applied to determine the location of the different functional RAF groups in the RAF

aggregates. Because of the flexibility of the hydrophobic tails of RAF, we anticipated that RAF forms micellar aggregates rather than face-to-face stacked aggregates[6]. The aggregation of surface-active molecules into micellar structures results in marked changes in the environment and in the dynamics of the molecules. These changes can be revealed by a number of NMR parameters, such as chemical shift, the relaxation rate, and the diffusion coefficient. These changes in the parameters have been be exploited to study the self-association process in micelles[19, 30]. For example, Tribolet et al. identified the functional groups involved in adenosine 5′-monophosphate self-association by determining the 1H NMR chemical shift as a function of the adenosine 5′-monophosphate concentration[31]. The H1 NMR peak assignment (Figure 4a) for RAF was accomplished using both 1D 1H NMR and 1H–1H correlated spectroscopy (COSY) techniques (Figure S1-3 and Table S2 in the supporting information). To investigate the position of the different functional groups of RAF in micelles, we studied the influence of RAF concentration on the chemical shift of different functional groups. Figure 4b presents the corresponding 1D 1H NMR spectra. For the functional groups located in the core of the micelles, we anticipated the largest change in the chemical shift. While all peaks shifted upfield with increasing RAF concentration, the most significant shifts were observed with peaks 3, 5, 6, and 7. We conclude that the protons associated with peaks 3, 5, 6 and 7 are in the hydrophobic core of the RAF micelle. In contrast, the protons associated with peaks 1, 2, and 4 are located on the surface of the micelle. This result suggests that the deprotonated salicylamide group functions as the hydrophilic head, while the 3-chloro-4-(4-chlorophenoxy) phenyl and diiodobenzene groups function as the hydrophobic tail. In an RAF micelle, the hydrophobic tail of adjacent molecules interact with each other through hydrophobic interactions, while the hydrophilic heads remain in contact with the surrounding medium. In contrast, the chemical shifts remained the same from 0.1–10 mg/mL when acetone was used as the solvent (Figure S4 in the supporting information).



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4.3

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RAF solubility behavior and mechanisms in 0.1 N NaOH solution/acetone cosolvent

The solubility of RAF in 0.1 N NaOH solution/acetone cosolvent system was measured in order to select an appropriate cosolvent composition to prepare the feed solution for spray drying. We also measured the solubility in the water–acetone cosolvent as a reference. The results are presented in Figure 5a (water– acetone) and Figure 5b (0.1 N NaOH solution/acetone). The log solubility of RAF in water/acetone cosolvent increased linearly with an increase in the volume fraction of acetone. These data fit well with the log-linear model derived by Yalkowsky and Roseman (Equation 3) for correlating the cosolvent composition and the nonpolar compound’s solubility[32]. log = !" # 1 − !% ................................................................................................ Equation 2 where f is the volume fraction of the organic solvent, S, So, and Sw stand for the solubility of the solute in cosolvent, in pure organic solvent, and in pure water, respectively. Rearranging Eequation 1 results in log = !" − !% # !& ......................................................................................... Equation 3 The experimental value (7.2) of the log-linear solubility profile obtained from the least squares regression agrees with the theoretical value (7.1) calculated from ( !/ − !0 . The solubility profile of RAF in 0.1N NaOH solution/acetone cosolvent demonstrates a complex pattern. As shown in Figure 5b, the log solubility of RAF in 0.1 N NaOH solution/acetone cosolvent, as a function of acetone volume fraction, consists of three different regions: In region 1, where acetone content was below 30%, v/v, the log RAF solubility increased linearly with the increase in acetone fraction. In region 2, where the acetone content was between 30% and 70%, v/v, the log RAF solubility decreased linearly with the increase in acetone fraction. In stage 3, where acetone was above 70%, v/v, the log solubility increased with the increase in acetone fraction. Assuming that the self-association of unionized RAF is negligible and assuming the protonation of the anionic self-associated RAF is negligible due to the high pH (i.e., pH 13), there are three equilibria in 0.1


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N NaOH solution/acetone cosolvent system: (1) solubilization of RAF, (2) ionization of RAF, and (3) self-association of ionized RAF, and they are illustrated as follows[3]. 67

123/4 5 899: 123/4; /< ................................................................................ Solubilization (Equilibrium 1) =>

12 899: 1 ? # 2 @ ............................................................................................... Ionization (Equilibrium 2) =B

A1 ? 899: 1

Self-Association of Rafoxanide in Aqueous Media and Its Application

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