Investigating the Association Mechanism between Rafoxanide and

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Investigating the Association Mechanism between Rafoxanide and Povidone Fan Meng, Zhifeng Jing, Rui Ferreira, Pengyu Ren, and Feng Zhang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03174 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Investigating the Association Mechanism between Rafoxanide and Povidone

Fan Menga, Zhifeng Jingb, Rui Ferreirac, Pengyu Renb, and Feng Zhanga,*

a

Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, the University

of Texas at Austin, 2409 University Avenue, Austin, Texas, USA b

Biomedical Engineering, the University of Texas at Austin, 107 W. Dean Keeton Street,

Austin, Texas, USA c Hovione

*To

LLC, 40 Lake Drive, East Windsor, New Jersey, USA

whom correspondence should be addressed

Department of Pharmaceutics College of Pharmacy, University of Texas at Austin 2409 University Avenue, A1920 Austin, Texas 78712 Phone: 512-471-0942 Fax: 512-471-7474 E-mail: [email protected]

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ACS Paragon Plus Environment

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ABSTRACT The low aqueous solubility of most hydrophobic medications limits their oral absorption. An approach to solve this problem is to make drug-polymer association. Herein, we investigated the association between rafoxanide, a surface-active, poorly water-soluble drug, with a commercial hydrophilic polymer povidone. We found that the association is a function of medium composition and could only take place in polar media, such as water. The association is favored by the hydrogen bond formation between the amide group in rafoxanide and the carbonyl group in povidone. In addition, the association is also favored by rafoxanide self-association through 𝜋𝜋 interaction between the benzene rings in adjacent rafoxanide molecules. 2D NMR has been applied to investigate the interactions and confirmed our hypotheses. Geometry optimization confirmed that RAF exists primarily in the antiparallel configuration in the rafoxanide aggregates. This study provides critical information for designing suitable drug-vehicle complex, and engineering the interactions between them to maximize the oral absorption. Our results shed lights on drug design and delivery, drug molecule structure-functionality relationship, as well as efficacy-enhancement toward interaction engineering. KEYWORDS oral bioavailability, poorly water soluble drug, amorphous solid dispersion, hydrogen bonding, hydrophobic interaction

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INTRODUCTION Pharmaceutical scientists are facing the challenges of drug delivery in R&D pipeline since 90% of these drugs are poorly water soluble.1 Low solubility in aqueous media could limit these drugs’ oral absorption and require formulation vehicles that are not suitable for some delivery routes. Various formulation strategies have been developed to improve apparent aqueous solubility and dissolution behavior of poorly water-soluble drugs. Drug-cyclodextrin and drugsurfactant associations are commonly used formulation strategies.2-4 In drug-cyclodextrin or drug-surfactant complexes, the excipients consisting of a nonpolar cavity interior and a polar exterior surface function as a molecular cage to “load” drug and the complexes themselves are soluble in aqueous media.5 However, mechanistic understanding of association between a hydrophobic drug and a hydrophilic polymer have been rarely investigated or optimized for increasing aqueous solubility of the drug. Rafoxanide (RAF) and Povidone K25 (PVP) have been chosen as the model compound and polymer in this study, respectively (Figure 1). RAF is a salicylamide derivative used for veterinary parasite control.6 The solubility of RAF in water is less than 0.1 μg/mL and only increases to 11 μg/mL in a 0.1 N NaOH solution. Thus, oral absorption of RAF is very limited. Several formulation strategies have been developed to improve the apparent aqueous solubility of RAF.7 Lo et al.8 reported that RAF forms a water-soluble complex with PVP in a co-solvent consisting of diluted NaOH solution in water and water-miscible organic solvents (e.g. acetone). The complex is formed in situ and remains in aqueous solution even after the removal of organic solvent. Through association with PVP, RAF in 0.1 N NaOH solution in water can reach a concentration of 75 mg/mL, which is about a 7,000-fold increase in solubility. However, the mechanisms of RAF-PVP association have not yet been studied and are poorly understood.

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Figure 1. Chemical structure of rafoxanide (RAF), povidone K 25 (PVP), salicylanilide and 1-ethyl-2-pyrrolidone

Association between PVP and several aromatic compounds such as salicylamide derivatives in various solvent systems has been investigated by Plaizier-Vercammen in the 1980’s.9-12 It was claimed that both hydrogen bond and hydrophobic interaction were involved in the association between salicylamide and PVP. In their study, the thermodynamics of association was studied using an ultrafiltration method. The association pattern was deduced from structure analysis and some thermodynamic values. As a derivative of salicylamide, RAF has some unique properties because of its long hydrophobic tail containing two ether-linked benzene rings. LogD of salicylamide and RAF at pH 6.8 is 0.74 and 6.70, respectively. Due to the presence of this hydrophobic tail, RAF was found to be surface active and aggregate into micelles in aqueous environment.13 Because of the self-association of RAF, the association between RAF and PVP is anticipated to be a lot more complicated. To put things in the right perspective, the percentage of salicylamide bound to PVP (at 4% concentration) ranged from 10 to 35%, depending on the ionic strength and pH.11 For RAF, more than 99% was bound to PVP (at 4% concentration). The objective of this study is to understand the thermodynamics and molecular mechanisms of the RAF and PVP association in two co-solvents: (1) 70% 0.1 N NaOH in water and 30% acetone, and (2) 20% ethanol and 80% acetone. Various techniques including isothermal titration 4


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calorimetry and solution NMR were used in this study. Computer simulation and modeling was also applied to study molecular interactions. The outcome from this study will help explain the interesting dissolution behaviors observed with RAF-PVP amorphous solid dispersions prepared using two different co-solvents. The release of RAF-PVP amorphous solid dispersion prepared using 70% 0.1 N NaOH in water-30% acetone in 50 mM phosphate buffer pH 6.8 was almost 20 times faster than that prepared using 20% ethanol-80% acetone. RAF also remained supersaturated in the dissolution medium and did not precipitate even after 2 weeks of storage at ambient condition. MATERIALS AND METHODS Materials Rafoxanide was purchased from ShenZhen Nexconn Pharmatechs Ltd. (ShenZhen, China). Kollidon K25 N.F. was kindly donated by BASF Chemical Company (Florham Park, NJ, USA). Salicylanilide and 1-ethyl-2-pyrrolidone were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). HPLC grade acetonitrile was purchased from Fisher Scientific (Pittsburgh, PA, USA). All other chemicals used in this study were ACS grade. Methods Rafoxanide Solubility as a function of PVP concentration The equilibrium solubility of RAF was determined at ambient condition (25°C). Excess amounts of RAF were added to vials that contained 10 mL of 0.1 N NaOH solution and various PVP concentration (0%, 1%, 2%, 5%, and 10%). After constant mixing using a magnetic stir bar for 48 h, the content was centrifuged at 1400g force using a Microfuge 18 Centrifuge (Beckman Coulter, Brea, CA, USA) for 10 min. 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. Isothermal Titration Calorimetry ITC measurement was performed using a Nano ITC (TA Instruments, New Castle, DE, USA). The reaction cell (V=1.34 mL) was filled with 20 mg/mL PVP solution in a co-solvent 5


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containing 70% 0.1 N NaOH solution and 30% acetone. The syringe (250 µL) was loaded with 50mg/mL RAF solution in the same co-solvent. The two solutions were mixed by injecting 10µL aliquots into the reaction cell at 25 °C. The injection-stirrer syringe was rotated at constant speed (250 rpm) throughout the experiment. Data from the injection of 50mg/mL RAF solution to pure co-solvent was acquired as reference. Nuclear Magnetic Resonance Spectroscopy 1H

NMR spectra were acquired on a Varian NMR 600 MHz Spectrometer (Agilent Inc., Palo

Alto, CA, USA) at 25 °C using standard sequences. RAF-PVP mixtures at 1:3 wt. ratio were dissolved with a RAF concentration of 5 mg/mL in fresh deuterated co-solvents. Two co-solvent systems were selected: co-solvent A contains 70% 0.1 N NaOH solution and 30% acetone; cosolvent B contains ethanol and 80% acetone. 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. 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. Double-stimulated echo pulse program was used with bipolar gradient pulses and a longitudinal eddy current delay. The relaxation delay was set at 2 s, the finite pulse decays were collected into 64,000 complex data points, and 64 scans were acquired for each sample. The gradient pulse strength was increased from 5% to 95% of the maximum strength of 60 G cm−1 in 50 steps. The squared gradient pulse strength was linearly distributed. For NOESY, the mixing time was set to 300 ms. Molecular Modeling of Rafoxanide Self-Association and Rafoxanide-PVP Association Quantum mechanical (QM) calculations were carried out at the HF/6-31G(d) level of theory using the Gaussian 09 software (Gussian, Inc. Wallingford, CT, USA). In geometry optimization, the solvent effect was taken into account by applying a SCRF (self-consistent reaction field) through the polarizable continuum model (PCM). Frequency analysis of each compound was performed at the same level to achieve the optimized minima/zero imaginary frequency. Molecular dynamics (MD) simulations were performed with the general Amber force field using AMBER 16 software (AMBER Software, San Francisco, CA, USA). In the calculation system, 6


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18-20 RAF molecules were placed in a cubic box of cosolvent consisting of 70% 0.1 N NaOH solution in water and 30% acetone mixture. The molar ratio between RAF and PVP monomer was 1:15, corresponding to the experimental weight ratio of 1:3. To accelerate the equilibration, 100-ns NVT simulations at 600 K were performed. Each structure extracted from the 600 K simulations was used as starting configuration for 10-ns cooling followed by 100-ns equilibrium simulations at 300 K. RESULTS AND DISCUSSION Enhanced Solubility of RAF in the Presence of PVP The chemical structure of RAF and its critical physicochemical properties are presented in Figure 1

and Table 1, respectively. The drug substance RAF is a poorly water-soluble weak acid

with a Log D of 6.70 (pH 6.8). Aqueous solubility of RAF is less than 0.1 μg/mL at pH 7, and it only reaches 10.7 μg/mL at pH 13 (0.1 N NaOH solution). The low aqueous solubility of RAF is mainly attribute to three hydrophobic benzene rings and intramolecular hydrogen bonding.14 In the current study, it was observed that the apparent aqueous solubility of RAF increased drastically in the presence of PVP. Figure 2 presents the apparent aqueous solubility of RAF in 0.1 N NaOH solution (pH 13) at different concentrations of PVP. The apparent solubility of RAF increased by 238, 568, 1263, and 2019 folds at 1%, 2%, 5% and 10% PVP, respectively. The drastic change in RAF solubility implied the formation of a water-soluble complex between RAF and PVP in 0.1 N NaOH solution in water. However, RAF-PVP association in aqueous media is difficult to characterize due to the poorly aqueous solubility of RAF. Therefore, RAF-PVP association was investigated in a co-solvent containing 70% 0.1N NaOH solution in water and 30% acetone mixture to simulate the association in aqueous solution.

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Table 1. Critical physicochemical properties of RAF and PVP

Physicochemical properties Molecular weight Melting point a Melting enthalpy a Glass transition temperature a LogD b pKa c

RAF 626 175.4 ºC 61.12 J/g 61.6 ºC 6.7 4.6

PVP 23,000-25,000 N/A N/A 121.1°C N/A N/A

measured using differential scanning calorimetry measured at pH 6.8 phosphate buffer c measured using UV method a

b

Thermodynamics of RAF-PVP association Isothermal titration calorimetry (ITC) was used in the current study to investigate the thermodynamics of RAF-PVP interactions in two co-solvents. ITC is a sensitive technique to detect the enthalpy changes resulted from the molecular interaction during the titration of one ligand solution to another ligand solution. ITC has been applied to study a wide range of molecular interaction including small molecule-protein, polymer-polymer, and drug-polymer interactions.15-18 A complete set of thermodynamic parameters of interaction including dissociation constants, enthalpy, entropy, and free energy can be derived from ITC experiment. RAF solubility (mg/mL)

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30 25 20 15 10 5 0 0.00

0.05

0.10

0.15

PVP% (w/v) Figure 2. The effect of Povidone K25 concentration on the apparent solubility of rafoxanide in 0.1 N NaOH solution.

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The ITC thermogram of RAF-PVP association in a 70% 0.1 N NaOH in water-30% acetone mixture is presented in Figure 3. An average PVP molecular weight of 24,000 was used for the calculation. It was concluded that the association is spontaneous since free energy change (-15.3KJ.mol-1) is negative. The dissociation constant (Kd) was determined to be 1.436x10-3 M. This value is similar to that of the association between PVP and some aromatic compounds in water. As an example, the dissociation constant between salicylamide and PVP in boric acidsodium hydroxide buffer pH 9.2 was measured to be 4.762x10-4 M, with a free energy change of -18.8KJ.mol-1.11 The binding stoichiometry (n=17.31) indicated that the average molar ratio between RAF molecule and the PVP polymer was 17.31:1. In other words, the weight ratio between RAF and PVP is 1:2.2. According to these thermodynamic parameters, RAF-PVP association was both enthalpically (negative) and entropically (positive) favorable. It was hypothesized that the decrease in enthalpy was due to the exothermic reaction resulting from the hydrogen bonding (hydrophilic interaction). The increase in entropy suggested the presence of hydrophobic interactions including the self-association of RAF molecules and RAF-PVP hydrophobic interactions. Surface-active properties and self-association of RAF in 70% 0.1 N NaOH in water-30% acetone mixture was reported in our previous study.13 The notion of the increase in the overall entropy is counterintuitive, since the association of RAF with PVP reduces the disorder of both RAF and PVP molecules. As shown in Figure 4, the “clathrate water cage” around RAF and PVP was disrupted when RAF complexed with PVP. This disruption increased the disorder of these water molecules, and hence increased the overall entropy. This concept was firstly introduced by Kauzmann to explain the hydrophobic interaction in aqueous media.19 Both hydrophilic hydrogen bonding interaction and hydrophobic interaction were confirmed using various NMR techniques and the results are presented in the next section.

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Figure 3. Isothermal titration calorimetry thermogram of rafoxanide and povidone K25 in 70% 0.1N NaOH in water-30% acetone mixture at 25°C, 50 mg/mL RAF solution was titrated into 20 mg/mL PVP solution.

In contrast, no change in enthalpy was observed when a 20% ethanol-80% acetone mixture was used as the titration medium. It was concluded that RAF and PVP did not interact in this organic cosolvent. Molecular Mechanisms for RAF-PVP Association in 70% 0.1 N NaOH- 30% Acetone Mixture The molecular mechanisms for association between RAF and PVP in these two co-solvent systems were investigated using various NMR techniques. As indicated by the ITC data, the strong association in a 70% 0.1 N NaOH in water-30% acetone mixture was attributed to hydrophilic interaction (hydrogen bonding) and hydrophobic interactions (self-association of RAF, and potentially hydrophobic interaction between RAF and PVP). RAF and PVP did not interact in a 20% ethanol-80% acetone mixture, since hydrophobic interactions could not take place in that medium.13 A detailed discussion of the molecular mechanisms of the interactions is presented in the following sections.

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Figure 4. Diagram to illustrate water cage around rafoxanide and release of water following rafoxanide association.

Hydrogen bonding between RAF and PVP Hydrogen bonding between drugs and povidone has contributed to the enhancement of drug solubility.13 Povidone functions as a hydrogen bond acceptor, while drugs are hydrogen bond donors. RAF and PVP could interact via hydrogen bonds. In a 70% 0.1 N NaOH in water-30% acetone mixture, the phenol group (O-H) is ionized, and only the amide group (N-H) of RAF can function as hydrogen bond donor. In a 20% ethanol-80% acetone mixture, both phenol (O-H) and amide (N-H) groups are unionized and can act as hydrogen bond donors. 1H NMR technique was applied to definitively determine whether hydrogen bonding plays a role in RAF and PVP association. If hydrogen bonding is present, the chemical shift of neighboring protons would change due to the change in electron density and magnetic induction effect.20-22 1D 1H NMR spectra of RAF or NaRAF in the presence or absence of PVP in two media are shown in Figure 2. The assignment of 1H NMR peaks for RAF was accomplished in our previous study using a combination of 1D 1H NMR and 1H−1H correlated spectroscopy (COSY) techniques.13 The 1H NMR spectrum of RAF in 70% 0.1 N NaOH solution in H2D-30% acetone mixture is presented as Figure 5-A2.

When PVP was added, downfield shifting and line

broadening of 1H NMR peaks (1, 2, and 4) for protons near the amide group confirmed the formation of hydrogen bond between the amide group (N-H) of RAF and the carbonyl group (C=O) of PVP. The electron withdrawing by the carbonyl group lowered the electron density 11


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around the amide group in RAF and therefore less shielding. As a result, NMR signals for protons near the amide group shifted downfield. Broadening of NMR signals was due to the association of RAF with PVP. The width of NMR signal is proportional to the reciprocal of T2 relaxation (Equation 1). 1

𝑣1/2 = 𝜋𝑇2 .........................................................................................................................Equation 1 where 𝑣1/2 is the width of NMR signal at half height and T2 is spin-spin relaxation time. The association of RAF (a small molecule with a molecular weight of 626) with PVP (a polymer with a molecular weight of 24,000) slowed down the tumbling motion of RAF, and hence shortened T2 relaxation (spin-spin relaxation).23, 24 As a result, broader NMR signals were observed in the presence of PVP. In contrast, neither shift nor broadening of NMR signals was observed with a 20% ethanol-D680% acetone-D6 mixture as the medium, indicating the absence of specific interactions between RAF and PVP in that cosolvent (Figure 5, B1 and B2). This result agreed well with the observation from ITC study. There are three hypothetical molecular mechanisms for the observed difference in RAF-PVP interactions in two different media: (1) different RAF ionization states. RAF is in ionized and unionized forms in a 70% 0.1 N NaOH in water-30% acetone and a 20% ethanol-80% acetone mixtures, respectively; (2) different acetone volume ratios. In a 20% ethanol-80% acetone mixture, acetone is the dominant component, it can act as a hydrogen bond acceptor and compete with PVP for hydrogen bonding with RAF; and (3) different aggregation states of RAF. Our previous study demonstrated that RAF self-associates in 70% 0.1 N NaOH in water-30% acetone but not in 20% ethanol-80% acetone mixture.

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Figure 5. 1H NMR spectra of rafoxanide (RAF) or its sodium salt (NaRAF) at 5 mg/mL in the presence or absence of Povidone K25 (PVP) at 15 mg/mL in deuterated aqueous or organic cosolvents. (A1) RAF and PVP in aqueous cosolvent, (A2) RAF in aqueous cosolvent, (B1) RAF and PVP in organic cosolvent, (B2) RAF in organic cosolvent, (C1) NaRAF and PVP in organic cosolvent, and (C2) NaRAF in organic cosolvent. Aqueous cosolvent: 70% 0.1 N NaOH in D2O-30% acetone-D6 mixture; organic cosolvent: 20% ethanol-D6-80% acetone-D6.

To evaluate the first hypothesis, we prepared the sodium salt of RAF (NaRAF) and conducted 1H NMR analysis of NaRAF in a 20% ethanol-80% acetone mixture with and without PVP. As shown in Figure 5-C1 and C2, there was no chemical shift or peak broadening with the addition of PVP. It was concluded that the difference in drug-polymer interactions in two media was not attributed to different ionization states of RAF. However, this does not mean that ionization of RAF may not be required for RAF and PVP to complex. Our previous study found that RAF self-associates only when ionized.)

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To evaluate the second hypothesis, we studied the 1H NMR spectra of RAF in chloroform with and without PVP. Chloroform was selected because it is a non-polar solvent without hydrogen bond accepting or donating capability. As shown in Figure 6, there was no chemical shift or peak broadening when PVP was added. This result confirmed that different RAF-PVP interactions in the two co-solvents were not due to the difference in the acetone volume percentage. Evaluation of the third hypothesis is discussed in the next section.

Figure 6. 1H NMR spectra of various solution; rafoxanide in chloroform-D1 with Povidone K25 (A1) or without Povidone K25 (A2); Salicylanilide in 70% 0.1N NaOH in D2O- 30% acetone-D6 mixture with Povidone K25 (B1) or without Povidone K25 (B2); rafoxanide in 70% 0.1N NaOH in D2O- 30% acetone-D6 mixture with 1-ethyl-2pyrrolidone (C1) or without 1-ethyl-2-pyrrolidone (C2).

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Hydrophobic interactions between RAF molecules in RAF-PVP complex In our previous study, RAF was observed to be surface-active and to self-aggregate in a 70% 0.1 N NaOH-30% acetone mixture, but not in a 20% ethanol-80% acetone mixture. Both the hydrophilic head (2-hydroxy-3,5-diiodobenzamide) and hydrophobic tail (3-chloro-4-(4chlorophenoxy) phenyl) groups of RAF were identified. We believed that self-aggregate of RAF in RAF only solution was driven by the hydrophobic 𝜋-𝜋 stacking of aromatic rings in the hydrophobic tail.13 In the current study, whether there exists hydrophobic interaction between RAF molecules in RAF-PVP complexes formed in a 70% 0.1 N NaOH-30% acetone mixture was investigated. It was hypothesized that hydrophobic interaction between RAF molecules played an important role in RAF-PVP association.

Figure 7. RAF-RAF region in 2D NMR NOESY spectra of rafoxanide (5 mg/mL) and Povidone K25 (15 mg/mL) mixture in (A) 70% 0.1N NaOH in D2O-30% acetone-D6 mixture and (B) 20% ethanol-D6-80% acetone-D6 mixtures.

2D NMR NOESY is a common technique to assess molecular interactions in solution state. Cross peaks in NOESY spectra connect resonances from nuclei that are spatially close to each other, and hence are considered definitive signs for physical interactions.25-27 For small molecules, cross-peaks can be observed between protons that are no greater than 4 Å apart, while the upper limit for large molecules is about 5Å. NOESY spectra of RAF-PVP are presented in Figure 7.

In a 20% ethanol-D6-80% acetone-D6 mixture (Figure 7B), cross-peaks observed for H4-

H6 and H5-H7 were due to intramolecular coupling. In a 70% 0.1 N NaOH in D2O-30% acetone15


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D6 mixture (Figure 7A), cross-peaks were observed between H5/H7 and all other protons, indicated 𝜋-𝜋 stacking between different benzene rings. In fact, 𝜋-𝜋 stacking is a typical hydrophobic interaction and is driven by the desolvation of the aromatic faces.

28-30

General

speaking, the strength of 𝜋-𝜋 stacking increases with increasing solvent polarity.30 Different stacking patterns, such as parallel staking, T-shape staking and edge-to-face stacking, can be formed between aromatic rings.31 The 𝜋-𝜋 stacking patterns in RAF self-aggregates are discussed in the next section. To study the significance of hydrophobic interactions of RAF molecules in RAF-PVP association, the interaction between salicylanilide and PVP was compared against that between RAF and PVP. Salicylanilide and RAF have identical hydrophilic head with RAF except for two iodine substitutes at the meta position. However, the size of the hydrophobic tails is significantly different. There are one and two benzene rings in salicylanilide’s and RAF’s hydrophobic tails, respectively. The 1H NMR spectra of salicylanilide in the presence and absence of PVP in 70% 0.1 N NaOH in D2O-30% acetone-D6 mixture are shown in Figure 6. NMR result confirmed that salicylanilide did not aggregate in a 70% 0.1 N NaOH in D2O-30% acetone-D6 mixture (Figure 6-B1). Neither change in chemical shift nor line broadening was observed for salicylanilide protons when PVP was added (Figure 6-B2). It was concluded that salicylanilide did not interact with PVP in a 70% 0.1 N NaOH in D2O-30% acetone-D6 mixture. This result confirmed our third hypothesis that the hydrophobic interaction of RAF was essential to RAFPVP association. Self-aggregation of RAF molecules due to the hydrophobic interactions was confirmed via molecular modeling, which is presented in a later section of this manuscript. The importance of RAF self-aggregation due to the hydrophobic - interactions in enhancing RAF-PVP association could also be explained from a thermodynamic perspective. RAF aggregates facilitate its association with PVP since the RAF in the aggregated state is more orderly. As a result, the loss of entropy due to the RAF-PVP association is less for RAF aggregates than that that for free RAF. The phenomenon that molecules in an aggregated state (physically or chemically) have a higher tendency to interact than those in a free state is well recognized in the field of inter-polymer 16


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association.

32, 33

Poly(acrylic acid) and poly(vinyl pyrrolidone) can complex and precipitate out

of aqueous solution, while their respective monomers do not interact with each other.34 To further illustrate the effect of the aggregation state on molecular interactions, we studied the interaction between RAF and 1-ethyl-2-pyrrolidon, the repeating unit of PVP. As shown in Figure 6 (C1 and C2), neither change in chemical shift nor line broadening was observed for RAF protons when 1-ethyl-2-pyrrolidone was present. Hydrophobic interactions between RAF and PVP Besides hydrophobic - interaction between RAF molecules, hydrophobic interaction between RAF and PVP was also observed in a RAF-PVP complex. Figure 8 presents the regions of RAF (X-axis) to PVP (Y-axis) in 2D NMR NOESY spectrum. Cross-peaks were observed for protons in benzene ring III of RAF (H5 and H7) and protons in the 5-membered lactam of PVP. These cross-peaks provided a strong evidence for the close proximity between RAF and PVP, and the hydrophobic interaction between benzene ring III of RAF and the 5-membered lactam ring of PVP.

Figure 8. RAF-PVP region in 2D NMR NOESY spectra of rafoxanide (5 mg/mL) and Povidone K25 (15 mg/mL) mixture in (A) 70% 0.1N NaOH in D2O-30% acetone-D6 mixture. X-axis represents rafoxanide and Y-axis represents povidone.

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Simulation and Modeling of RAF and PVP Interactions Computational modeling has become a vital adjunct to experimental studies on the mechanisms of molecular interactions.35-37 Modeling is commonly used to compute the energy of a particular molecule structure, perform geometry optimization and simulate molecular dynamics.38, 39 In the current study, the molecular mechanisms for RAF self-aggregation and RAF-PVP association in 70% 0.1 N NaOH solution in water and 30% acetone cosolvent were investigated using quantum mechanical (QM) and molecular dynamics (MD) modeling, respectively. QM analysis was used to explore the possible stacking structures of RAF aggregates in RAF only solution. Geometry optimization, which identifies the lowest-energy molecular structure in close proximity, was performed.38 Figure 9 presents four possible structures obtained from QM geometry optimization with different starting structures. Specifically, structures in Figure 9c and d are in the lowest energy state. This result is consistent with that from 2D NMR NOESY analysis (Figure 7A). Both of these methods demonstrated that protons in different benzene rings from the neighboring RAF molecules are in close proximity. The result from geometry optimization also confirmed that RAF exists primarily in the antiparallel configuration in the RAF aggregates.

Figure 9. Optimized structures of RAF dimers: parallel stacking (a), open conformation (b) and anti-parallel stacking (c and d) in 70% 0.1 N NaOH solution in water and 30% acetone mixture.

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Molecular dynamics calculations were applied to explore the possible configurations of the RAFPVP complex in 70% 0.1 N solution in water and 30% acetone mixture. The spatial arrangement of molecules is illustrated in Figure 10. Even though the limited system size in the simulations prevented the formation of large RAF aggregates, RAF aggregation was clearly observed. Both hydrogen bonding and hydrophobic 𝜋-𝜋 stacking are involved in the RAF-PVP association. Antiparallel stacking between RAF molecules is illustrated in Figure 10A. Face-to-edge stacking for benzene rings I-III and I-II is illustrated in Figure 10B. Apart from 𝜋-𝜋 stacking, hydrogen bonding was also observed (Figure 10C). The distance between the carbonyl oxygen of PVP and the amide proton of RAF was calculated to be 2.91Å. The interaction patterns simulated from MD calculations agreed with the experimental data.

Figure 10. MD modeling of RAF-PVP system in 70% 0.1 N NaOH in water-30% acetone mixture. Anti-parallel stacking was observed between different RAF molecules.

CONCLUSIONS In summary, association between RAF and PVP was thoroughly investigated and the association mechanism was elucidated. The association is dependent on the media composition. In organic co-solvent consisting of 20% ethanol and 80% acetone, association did not occur. In contrast, strong association was observed in aqueous cosolvent consisting of 70% 0.1 N NaOH in water and 30% acetone. The association was found to be both enthalpically (-1.223 KJ.mol-1) and entropically (50.32 J.mol-1.K-1) favored. In addition, the dissociation constant was 1.436x10-3 M. 19


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According to 2D NOESY NMR experimental data and analysis of the radial distribution, the association was driven by both hydrophilic hydrogen bonding between RAF and PVP, and two hydrophobic interactions including (1) RAF-RAF self-association and RAF-PVP hydrophobic interaction. RAF-RAF self-association due to hydrophobic 𝜋-𝜋 interactions was further verified by the simulation and modeling. Besides, RAF aggregates primarily existed in the antiparallel configuration according to the modeling. The simulation also showed that both RAF monomer and aggregate bind to PVP polymer chains. These findings not only provide examples for designing drug-polymer complex but also understanding the interactions between them on molecular level. Our research will draw attention from pharmaceutic and drug scientists and will evoke more researches on the molecular design and potency-driving mechanism study.

CONFLICT OF INTEREST There are no conflicts to declare.

ACKNOWLEDGEMENT The authors want to thank Yongcao Su, Principle Scientist at Merck, for the technical discussion of NMR results.

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TABLE OF CONTENTS GRAPHIC

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Investigating the Association Mechanism between Rafoxanide and

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