Molecular complexation of curcumin with pH sensitive cationic copolymer enhances the aqueous solubility, stability and bioavailability of curcumin

    Molecular complexation of curcumin with pH sensitive cationic copolymer enhances the aqueous solubility, stability and bioavailability of curcumin Sunny Kumar, Siddharth S. KesharwaniHimanshi Mathur, Mohit Tyagi, G. Jayarama Bhat, Hemachand Tummala PII: DOI: Reference:

S0928-0987(15)30063-4 doi: 10.1016/j.ejps.2015.11.010 PHASCI 3406

To appear in: Received date: Revised date: Accepted date:

24 August 2015 10 November 2015 11 November 2015

Please cite this article as: Kumar, Sunny, KesharwaniHimanshi Mathur, Siddharth S., Tyagi, Mohit, Jayarama Bhat, G., Tummala, Hemachand, Molecular complexation of curcumin with pH sensitive cationic copolymer enhances the aqueous solubility, stability and bioavailability of curcumin, (2015), doi: 10.1016/j.ejps.2015.11.010

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ACCEPTED MANUSCRIPT Molecular Complexation of Curcumin with pH Sensitive Cationic Copolymer Enhances the Aqueous Solubility, Stability and Bioavailability of Curcumin

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Hemachand Tummala

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Sunny Kumar Siddharth S. Kesharwani Himanshi Mathur , Mohit Tyagi , G. Jayarama Bhat

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Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University,

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SAV # 255, Box 2202C, Brookings, South Dakota 57007, United States

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* Corresponding author:

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Associate Professor

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Department of Pharmaceutical Sciences

College of Pharmacy, South Dakota State University SAV # 255, Box 2202C

Brookings, South Dakota 57007, United States Phone no. +1-605-688-4236 Fax +1-605-688-5993 E-mail: [email protected]

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ACCEPTED MANUSCRIPT ABSTRACT Curcumin is a natural dietary compound with demonstrated potential in preventing/treating

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several chronic diseases in animal models. However, this success is yet to be translated to

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humans mainly because of its poor oral bioavailability caused by extremely low water solubility. This manuscript demonstrates that water insoluble curcumin (~1µg/ml) forms highly aqueous

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soluble complexes (>2mg/ml) with a safe pH sensitive polymer, poly(butyl-methacrylate-co-(2-

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dimethylaminoethyl) methacrylate-co-methyl-methacrylate) when precipitated together in water. The complexation process was optimized to enhance curcumin loading by varying several

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formulation factors. Acetone as a solvent and polyvinyl alcohol as a stabilizer with 1:2 ratio of drug to polymer yielded complexes with relatively high loading (~280µg/ml) and enhanced

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solubility (>2mg/ml). The complexes were amorphous in solid and were soluble only in buffers with pHs less than 5.0. Hydrogen bond formation and hydrophobic interactions between

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curcumin and the polymer were recorded by infrared spectroscopy and nuclear magnetic resonance spectroscopy, respectively. Molecular complexes of curcumin were more stable at various pHs compared to unformulated curcumin. In mice, these complexes increased peak plasma concentration of curcumin by 6 times and oral bioavailability by ~20 times. This is a simple, economic and safer strategy of enhancing the oral bioavailability of curcumin.

KEYWORDS: Curcumin, Molecular Complexation, Oral delivery, Eudragit® EPO, Solubility, Bioavailability, Stability

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ACCEPTED MANUSCRIPT 1. Introduction Curcumin, is a dietary polyphenolic compound with demonstrated beneficial effects in various

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complex chronic diseases such as cancer (Aggarwal et al., 2003; Aggarwal et al., 2007; Goel and

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Aggarwal, 2010; Ruby et al., 1995; Yallapu et al., 2012), atherosclerosis (Aggarwal and

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Harikumar, 2009), diabetes (Nishiyama et al., 2005; Sharma et al., 2006), Alzheimer’s disease (Lazar et al., 2013), inflammatory bowel syndrome (Aggarwal and Harikumar, 2009; Aggarwal

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et al., 2003; Rahman et al., 2006), arthritis (Gupta et al., 2013b), cystic fibrosis (Egan et al.,

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2004), malaria (Isacchi et al., 2012) etc. These are complex diseases with multiple etiologies that need interference at several signaling pathways. The unique advantage of curcumin stands

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because of broad spectrum of pharmacological activities it possesses that interferes at multiples

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pathways in the above-mentioned diseases (Aggarwal et al., 2007; Anand et al., 2007; Gupta et al., 2013a; Gupta et al., 2013b). More importantly, curcumin is generally recognized as safe

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(GRAS) by the United States Food and Drug Administration (US-FDA) for daily use (Basnet and Skalko-Basnet., 2011). Despite its high potential in treating these complex chronic diseases, the clinical advancement of curcumin as an oral therapy is hindered due to its low bioavailability after administration (% of administered drug reaching the blood circulation) and short biological half-life. This is mainly due to its poor water solubility (< 1 µg/ml), degradation in aqueous fluids and rapid metabolism in the body (Aggarwal et al., 2003; Anand et al., 2007; Cheng et al., 2000; Gupta et al., 2013a; Prasad et al., 2014). To overcome these pharmacokinetic challenges, impractically large doses of curcumin were administered orally in some clinical studies (2-4 g/day). This approach is practically not feasible for daily clinical application (Lao et al., 2006). Even administering very high therapeutic doses (10-12 g/day) of curcumin or a chronic oral

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ACCEPTED MANUSCRIPT administration of curcumin (1–4 g/day) for 6 months could not produce more than 300nM peak blood concentration in humans (Cheng et al., 2000; Lao et al., 2006).

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Various strategies to overcome the issue of poor water solubility, thus, associated low

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bioavailability have been investigated (Anand et al., 2007; Bansal et al., 2011; Prasad et al., 2014). These strategies include; structural modification (Anand et al., 2008; Ruby et al., 1995),

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chelation and bioconjugates (Gao et al., 2011; Pandey et al., 2011), use of adjuvants which block

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the curcumin metabolism (Pandey et al., 2011), formulating into complex dosage forms such as, micelles (Gou et al., 2011; Sahu et al., 2008), liposomes (Agarwal et al., 2013; Isacchi et al.,

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2012), polymeric nanoparticles (Basniwal et al., 2011; Dandekar et al., 2010; Yallapu et al., 2012), lipid nanoparticles (Li et al., 2012), cyclodextrin complexes (Yadav et al., 2010) etc.

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Although some of these strategies showed improvements in enhancing the bioavailability of curcumin, they have not translated into daily human use. This is due to high cost of the

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materials/formulations, toxicity associated with the formulations, and/or incompatibility for oral use. Therefore, there is an unmet critical need for a formulation that enhances the solubility and bioavailability of curcumin in a safe, economical, and practical for daily oral use for the above mentioned chronic diseases (Baum et al., 2008; Dhillon et al., 2008). Eudragit® EPO is a pH sensitive cationic copolymer of poly(butyl-methacrylate-co-(2dimethylaminoethyl) methacrylate-co-methyl-methacrylate). It is soluble in aqueous solutions with pHs less than 5.0. Eudragit® EPO is an US-FDA approved polymer for oral consumption in humans and importantly, it is very economical and safe for daily use. There have been numerous reports on the solid dispersions of various drugs with Eudragit® EPO to increase their solubility and bioavailability. Recently, Eudragit ® EPO has also been shown to interact with drugs

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ACCEPTED MANUSCRIPT through non-covalent interactions (Frank et al., 2012; Gangurde et al., 2015; Kerdsakundee et al., 2015; Kojima et al., 2012; Li et al., 2015; Liu et al., 2010; Meng et al., 2015).

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In this study, we have tested a novel strategy of forming molecular complexes of curcumin

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with Eudragit® EPO by co-precipitation. The complexes were characterized by various physicochemical methods. Curcumin formed complexes with the polymer through hydrophobic

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interactions. The complexes are stable even in aqueous solutions. These complexes enhanced

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increased its oral bioavailability in mice.

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solubility of curcumin by more than 20,000 times in aqueous buffers and more importantly,

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2. Materials and Methods

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2.1 Materials

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Synthetic Curcumin (>98% pure) and polyvinyl alcohol (PVA) were purchased from Fisher Scientific (Pittsburgh, PA, USA), Eudragit® EPO was a gift from Evonik Industries (Birmingham, AL, USA), acetone, ethyl acetate, ethanol and other solvents were purchased from Fisher Scientific (Pittsburgh, PA, USA), Tween-20 from Amresco (Solon, OH, USA), Pluronic F-68 from Panreac AppliChem (St. Louis, MO, USA). Spin-X® UF concentrator tubes with 10kDa cutoff membrane, Buffers and other biochemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). 2.2 Formation of Curcumin-Eudragit® EPO Molecular Complexes (CEMCs). CEMCs were prepared using nanoprecipitation method. Curcumin and Eudragit® EPO were dissolved in an organic solvent. The solution was then added drop-wise to an aqueous solution containing a 3% w/v of stabilizer under constant stirring (200 rpm). The resulting dispersion was stirred until the complete evaporation of organic solvent. Subsequently, the dispersion was 5

ACCEPTED MANUSCRIPT centrifuged to collect the formed complexes, freeze-dried and stored at 4 ˚C. Precaution was taken throughout the study to protect the curcumin from light and all the experimental

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procedures were undertaken in dark.

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2.3 Measurement of Curcumin Loading and Apparent Solubility in Aqueous Buffers.

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Curcumin loading (loading represents the micrograms of curcumin present in one milligram of CEMCs formulation) was determined by first extracting the curcumin from a known amount of

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CEMCs into ethanol. The amount of curcumin present in the ethanolic extract was measured at

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420 nm using an UV−visible spectrophotometer (Gangurde et al., 2015; Garcea et al., 2004; Kerdsakundee et al., 2015; Li et al., 2015). To determine the apparent solubility (apparent

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solubility represents the solubility measured at 4 h and not the solubility at equilibrium) of

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curcumin, unformulated curcumin or equivalent amount of CEMCs were dispersed in pH 1.2

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solution (0.1N HCl) and incubated at 37 °C for 4 h with 100 rpm shaking. After incubation, the samples were centrifuged at 20,000g, the supernatant filtered through 0.2 µM filter and subsequently analyzed for the concentration of curcumin by using UV absorbance at 420 nm. There was no interference on the absorbance at 420 nm by the excipients of the formulation, the polymer or PVA. Therefore, there was no need to separate them through chromatography before we detect curcumin. 2.4 Method Optimization. The process of CEMCs formation was optimized to enhance curcumin loading and its aqueous solubility by varying the type of organic solvent (acetone, ethyl acetate, and ethanol) and stabilizers/surfactants (tween-20, pluronic F-68, and polyvinyl alcohol (PVA) used to prepare the molecular complexes. The ratio of curcumin to Eudragit® EPO used in preparation of the

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ACCEPTED MANUSCRIPT molecular complexes was also varied from 1:2, 1:3 and 1:5 of curcumin respectively per 100 mg of Eudragit® EPO to further enhance the curcumin loading.

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Initially, CEMCs were prepared using tween-20 as surfactant with three different solvents

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(acetone, ethyl acetate and ethanol) and with 20 mg of curcumin per 100 mg of Eudragit® EPO.

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Subsequently, acetone was chosen for further studies and the type of stabilizers/surfactants (tween-20, Pluronic F-68 or PVA) was varied. Finally, CEMCs were prepared using different

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ratios of curcumin to Eudragit® EPO with PVA and acetone. Apparent solubility of curcumin

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from unformulated curcumin and CEMCs was measured by dispersing 2 mg equivalent of curcumin per ml of pH 1.2 solution and incubated at 37 ˚C for 4 h in dark with 100 rpm shaking.

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The curcumin dissolved after 4 h was analyzed by UV absorbance at 420 nm. There was no

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interference on the absorbance at 420 nm by the excipients of the formulation, the polymer or

curcumin.

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PVA. Therefore, there was no need to separate them through chromatography before we detect

2.5 Assessment of Curcumin-Eudragit® EPO Molecular Complex (CEMCs) Formation. Curcumin and CEMCs were dissolved in ethanol and 50 mM acetate buffer (pH 4.5 solution) respectively. The samples were centrifuged at 20,000g and the supernatant was collected and the concentration of curcumin was determined. Subsequently, the supernatant was passed through Spin-X® UF concentrator tubes purchased from Fisher Scientific (Pittsburgh, PA, USA) with 10 kDa cutoff membrane. The concentration of curcumin in the pass through was determined using an UV−visible spectrophotometer at 420 nm. 2.6 Aqueous Stability of Curcumin-Eudragit® EPO Molecular Complexes (CEMCs).

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ACCEPTED MANUSCRIPT The aqueous stability of soluble CEMCs was assessed at pH 1.2, 4.5, 6.5, or 7.4. Curcumin or the curcumin equivalent of CEMCs were dissolved in respective buffers containing 10 %

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methanol and incubated at 37 ˚C. At different time intervals, the samples were collected, centrifuged at 20,000g and the supernatant was passed through 0.2 μM filter. The amount of

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soluble and stable curcumin in the filtrate was determined by using an UV−visible

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spectrophotometer at 420 nm.

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2.7 Fourier Transform Infrared (FTIR) Spectroscopy.

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FTIR spectra of curcumin, Eudragit® EPO polymer, the physical mixture of curcumin and Eudragit® EPO, and CEMCs were performed using Nicolet 380 ATR-FTIR spectrophotometer (Thermo Electron Corp., Madison, WI). Data was acquired between 4000 cm−1 and 400 cm−1 at a

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scanning speed of 4 cm−1 and 50 scans. The average of 50 scans data was presented.

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2.8 Nuclear Magnetic Resonance (NMR) Spectroscopy. Solution 1H NMR spectra of CEMCs (Curcumin and Eudragit® EPO molecular complexes) were recorded on a Bruker 400 MHz NMR spectrometer. Briefly CEMCs were dissolved in D2O: acidic H2O (1:1) followed by addition of various proportions of DMSO-d6 to observe the decomplexation of the molecular complexes. Sample solution was transferred to a NMR tube and the spectra was recorded. 2.9 Differential Scanning Calorimetric (DSC) Analysis. Differential scanning calorimetric (DSC) analysis of curcumin, Eudragit® EPO polymer, the physical mixture of curcumin and Eudragit® EPO, and CEMCs were performed using TA Instruments Q200 Differential Scanning Calorimeter (TA Instruments, New Castle, DE, USA). Samples were weighed (equivalent to curcumin) and placed in sealed Tzero aluminum hermetic 8

ACCEPTED MANUSCRIPT pans. With liquid nitrogen as coolant, samples were scanned at 10 °C/min from -20 °C to 300 °C and thermograms were recorded (Mukerjee and Vishwanatha, 2009; Yallapu et al., 2010).

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2.10 Powder X-ray Diffraction (XRD).

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Powder X-ray diffraction measurements of curcumin, Eudragit® EPO polymer, the physical

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mixture of curcumin and Eudragit® EPO, and CEMCs were recorded using Rigaku powder xray diffractometer using Cu radiation, running at 40 kV and 44 mA. For this study, samples

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were mounted on double sided silicone tape and measurements were performed from 2 °C to 60

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°C at a scan speed of 4 °C/min and increments of 0.02 °C (Mukerjee and Vishwanatha., 2009). 2.11 Scanning Electron Microscopy (SEM).

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The surface morphology of CEMCs was investigated using a scanning electron microscopy (SEM, Model S-3400N, Hitachi, Japan) at an accelerating voltage of 10 kV (Kumar et al., 2014).

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The dry samples were mounted on metal holders using conductive double-sided tape and sputter coated with a gold layer for analysis (Agar Auto Sputter Coater, UK). 2.12 Oral Bioavailability Studies in Mice. BALB/C mice (n = 4-5 per group, 6 − 8 weeks old) purchased from Charles River laboratories

(Wilmington,

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USA)

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used for the oral bioavailability in mice.

Curcumin (150 mg/kg) or equivalent CEMCs were orally administered to fasting mice (8 h) in water as suspension. Blood samples from mice were collected through retro-orbital plexus at different time points (0.5, 1, 2, 4, 8, 12, and 24 h) after administering the formulations. The curcumin present in the mouse plasma was immediately extracted with acetone and the amount of curcumin was determined by reverse phase high-performance liquid chromatography (HPLC) method as explained below. However for determining curcumin levels from mouse plasma 9

ACCEPTED MANUSCRIPT samples, the authors needed to separate curcumin from other biological components by organic extraction and reverse phase HPLC technique before UV detection. Hence the authors have

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employed reverse-phase HPLC coupled with UV detector for estimation of the amount of

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curcumin. All animal experimentation was performed in compliance with regulations of the Institutional Animal Care and Use Committees (IACUC) of South Dakota State University,

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Brookings, SD, USA.

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2.13 Quantification of Curcumin by Reverse Phase HPLC.

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The curcumin present in the mouse plasma was immediately extracted with acetone and the amount of curcumin was determined by reverse phase high-performance liquid chromatography

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(HPLC) coupled with UV detector. Chromatographic separation was achieved using Symmetry®

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C18 column (150mm x 4.6mm, 5µm, Waters, USA) with an isocratic elution using mobile phase

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composed of acetonitrile and 1% w/v citric acid buffer (pH 3.0) (60:40 v/v ratio). The flow rate was set at 1 ml/min. Curcumin was detected using a UV detector at 420 nm with a sample volume of 50 µl per injection (Ma et al., 2007). 2.14 Statistical Analysis.

Experiments were performed in multiples of triplicates for proper statistical analysis. The results of the experiments are reported in terms of mean ± standard deviation (SD). Analysis of variance was performed using student’s t-test. 3. Results 3.1 Curcumin Forms Molecular Complexes with Eudragit® EPO. CEMCs were prepared by controlled precipitation of both the compounds in water. The formulations were dissolved in ethanol and aqueous buffers with pH 1.2, pH 4.5, and pH 7.4. 10

ACCEPTED MANUSCRIPT Curcumin only dissolved in ethanol, but not in aqueous buffers. However, curcumin from CEMCs dissolved in high concentrations in aqueous buffers with pHs< 5.0 as well as in ethanol.

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Physical addition of Eudragit® EPO blank particles to curcumin did not alter the solubility

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profile of unformulated curcumin (data not shown). Blank particles were prepared exactly in the same manner as CEMCs with all the excipients except curcumin.

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The formation of the molecular complexes was confirmed by passing the soluble

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curcumin through a 10 kDa cut-off filter unit and analyzing the curcumin concentration in the top portion before centrifugation and at the bottom portion of the tube after centrifugation (Fig.

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1). Curcumin with a molecular weight of 0.368 kDa, would easily pass through a 10 kDa cut-off membrane with the buffer. In contrast, Eudragit® EPO has a molecular weight of ~ 47 kDa, and

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that prevents it and any curcumin that had formed a complex with it from passing through 10 kDa cutoff membrane. The ratio of the concentration of curcumin in solution that had passed

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through 10 kDa cut-off membrane to the initial concentration of the solution was found to be approximately one for unformulated curcumin dissolved in 10 % ethanolic solution (data not shown) or in absolute ethanol (Fig. 1). The soluble curcumin present in the acidic solution after complexation with Eudragit® EPO did not filter through 10kDa cut off membrane in detectable amounts which, suggests that the curcumin had formed soluble complex with Eudragit® EPO. The CEMCs remained intact in aqueous solutions. 3.2 Fourier Transform Infrared (FTIR) Spectroscopy. FTIR spectroscopy was used to ascertain the formation of the CEMCs. Curcumin (in red), Eudragit® EPO polymer (in purple), the physical mixture of curcumin and Eudragit® EPO (in green) and CEMCs (in cyan), were recorded for FTIR (Fig. 2). The -OH band of curcumin was completely diminished in CEMCs which, suggests that there may be a formation of

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ACCEPTED MANUSCRIPT intermolecular hydrogen bond between the phenolic -OH group of curcumin and the -C=O group of Eudragit® EPO backbone. It is important to note here that these interactions only exist in

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solid state.

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3.3 Nuclear Magnetic Resonance (NMR) Spectroscopy.

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Solution 1H NMR spectra of CEMCs (Curcumin and Eudragit® EPO molecular complexes) was performed to ascertain the interaction pattern driving the CEMC (Curcumin and Eudragit® EPO

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molecular complex) formation. We investigated the interaction pattern between curcumin and

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Eudragit® EPO using 1H NMR. CEMCs were taken in 1 ml of D2O: acidic H2O mixture (1:1). To this solution various amount of DMSO-d6 was added to disrupt the interaction. The signal

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that represents the aromatic region (6.0-9.5 ppm) was broad in D2O: acidic H2O (1:1) and starts

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appearing sharp as DMSO-d6 was added to the same sample (Fig. 3). This indicates that the

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complexation involves strong hydrophobic interactions between aryl skeleton of curcumin and alkyl chain of Eudragit® EPO. With the addition of organic solvent like DMSO, the complex might have broken down into curcumin and the polymer that might have made curcumin peaks to appear sharp. The hydrogen bonding interaction between hydroxyl group of curcumin and Eudragit® EPO possibly identified by FTIR may not be a sole reason for complexation. 3.4 Differential Scanning Calorimetric (DSC) Analysis. The DSC curves of curcumin (Fig. 4A), Eudragit® EPO polymer (Fig. 4B), the physical mixture of curcumin and Eudragit® EPO (Fig. 4C), and CEMCs (Fig. 4D) were recorded. Curcumin (Fig. 4A) showed a sharp melting peak at 180.16˚C, indicating its crystalline nature. Eudragit® EPO polymer (Fig. 4B) exhibits a small peak at 50˚C, referring to the relaxation peak that follows the glass transition but no distinct melting point was observed that suggests the

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ACCEPTED MANUSCRIPT amorphous nature of the polymer. The physical mixture of curcumin and Eudragit® EPO showed (Fig. 4C) a sharp melting peak of curcumin at 177.01˚C and the relaxation peak of

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Eudragit® EPO. This indicates that in physical mixture curcumin is in crystalline form and

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Eudragit® EPO in amorphous form. However, such distinct melting point peak of crystalline

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curcumin and Eudragit® EPO in the complex form.

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curcumin was absent in CEMCs (Fig. 4D) suggesting a complete amorphous nature of both

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3.5 Powder X-ray Diffraction (XRD).

The powder X-ray diffraction patterns of curcumin showed characteristic sharp peaks indicative

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of crystalline nature of curcumin (Fig. 5A). Eudragit® EPO did not show distinct peaks in XRD

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because of it is existence in amorphous form (Fig. 5B). The peaks representing the crystalline

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nature of curcumin are visible in the physical mixture of synthetic curcumin and Eudragit® EPO (Fig. 5C). In contrast, curcumin in CEMCs did not show any distinct peaks, which, further confirms the existence of curcumin and the polymer in amorphous form in CEMCs (Fig. 5D). 3.6 Scanning Electron Microscopy (SEM). The surface morphology of the CEMCs, prepared by the controlled precipitation, was determined by SEM. Fig.6 illustrates a rough surface texture of CEMCs, no defined particles were observed. The appearance of the amorphous form of CEMCs as shown in Fig. 6 further points the existence of the complexes as amorphous and supports the observation made in differential scanning calorimetric and powder x-ray diffraction analysis. 3.7 Acetone and PVA in the Formulation Significantly Enhanced Curcumin Loading and Apparent Solubility. 13

ACCEPTED MANUSCRIPT The method of preparing CEMCs was optimized by altering the formulation parameters to enhance the loading of curcumin (loading represents microgram of curcumin present per

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milligram of CEMCs) and also to further increase the aqueous solubility. The formulation

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parameters altered were the type of organic solvent, stabilizer/surfactant and the drug to polymer

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ratio.

The solubility and loading depends on the interactions between curcumin molecules and

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curcumin with polymer molecules during co-precipitation. This depends on the polarity of the

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solvent used to re-precipitate/re-crystallize curcumin and the polymer together. The organic solvents were selected based on their dielectric constant value, which is an indicative of the

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polarity of a solvent (Ethyl Acetate, 6.02; Acetone, 21; Ethanol, 24.5; and Methanol, 33).

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Tween-20 was used as the surfactant in these studies. When acetone was used as the solvent, higher curcumin loading and apparent solubility (75.17 ± 0.89 µg/mg, 0.51 ± 0.02 mg/ml) was

Table 1

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achieved compared to ethyl acetate or ethanol as a solvent (Table 1).

Optimization of loading and apparent solubility (in 4 hrs) of curcumin from CEMCs prepared using three different organic solvents and stabilizers. Data represents mean ± standard deviation (n=3). * indicates variance is significant (p <0.05) as compared to sample 1 and 3. ǂ indicates variance is significant (p <0.05) as compared to sample 4.

Sample

Solvent

Stabilizer

No.

1

Ethyl Acetate

Tween-20

Curcumin:

Curcumin

Curcumin apparent

polymer

loading

solubility (mg/ml)

ratio

(µg/mg)

1:5

61.70 ± 2.92 *

0.45 ± 0.01 *

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ACCEPTED MANUSCRIPT Ethanol

Tween-20

1:5

49.28 ± 1.51*

0.40 ± 0.02*

3

Acetone

Tween-20

1:5

75.17 ± 0.89 *

0.51 ± 0.02 *

4

Acetone

Pluronic F-68

1:5

82.52 ± 2.92ǂ

0.54 ± 0.0ǂ

5

Acetone

PVA

1:5

149.24 ± 0.99 ǂ

0.81 ± 0.01 ǂ

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2

From the above studies acetone was selected as a solvent and the type of stabilizer was

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altered (Table 1). The loading and apparent solubility of curcumin was significantly improved

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with PVA compared Tween 20 or Pluronic F-68 (p <0.05). Loading of curcumin into the CEMCs was further enhanced by altering the curcumin to polymer ratio (Table 2). Acetone was used as

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an organic solvent and PVA. The highest curcumin loading was achieved when 50 mg of

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to 278.6 µg/mg.

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curcumin was used per 100 mg of Eudragit® EPO (1:2), which enhanced the curcumin loading

Effect of curcumin to polymer ratio on the loading of curcumin in CEMCs prepared using acetone PVA. Data represents mean±standard deviation (n=3). Curcumin: Eudragit® Curcumin loading (µg/mg) EPO ratio 1:5

149.24 ± 0.99

1:3

175.66 ± 0.88

1:2

278.61 ± 2.72

3.8 CEMCs Significantly Enhance the Aqueous Solubility of Curcumin.

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ACCEPTED MANUSCRIPT Once the preparation of CEMCs was optimized, the apparent solubility of complexes was investigated in diluted acidic water (0.1N HCl) with pH 1.2 for 4 h that represents the dissolution

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in gastric fluid after oral consumption. After 4 hrs, soluble curcumin was separated and estimated

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by UV−visible spectrophotometer at 420 nm. Increasing amounts of curcumin or its equivalent amounts of CEMCs was added to acidic solution to investigate the dose dependent increase in its

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aqueous solubility. The aqueous solubility of curcumin was tremendously enhanced when

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delivered as CEMCs up to 20 mg/ml (Fig. 7). This is more than 20,000 times in comparison to unformulated curcumin (~1 µg/ml). Physical addition of Eudragit® EPO blank particles to

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curcumin did not enhance the solubility of curcumin (data not shown). The data suggests that the phenomenon is not in-situ because of the presence of polymer or PVA; the complex needs to be

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formed prior to its addition into aqueous buffers. 3.9 The Solubility of CEMCs in Aqueous Vehicles Depends on pH.

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It is known that the solubility of Eudragit® polymers is pH dependent. Eudragit® EPO dissolves only in solutions with pH less than 5.0. To investigate whether the solubility of curcuminEudragit® EPO molecular complexes is also pH dependent, CEMCs were dissolved in aqueous buffers with different pHs (pH 1.2, pH 4.5 and pH 7.4). Similar to Eudragit® EPO, CEMCs were also soluble in solutions at pH 1.2 and 4.5, but poorly soluble at pH 7.4. In contrast, curcumin was practically insoluble at all three pHs (pH 1.2, pH 4.5 and pH 7.4) (Fig. 8). An interesting and very important observation to note with CEMCs is that once it was dissolved at pH 1.2 solution, adjusting the pH back to 7.0 did not precipitate curcumin or CEMCs complex (data not shown). This observation becomes critical for oral delivery of curcumin, where the stomach pH is 1-3 for dissolution of the formulation and the intestinal pH may increase up to 7.0 at the later parts of ileum.

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ACCEPTED MANUSCRIPT 3.10 CEMCs Enhance the Aqueous Stability of Curcumin. Once the concern related to solubility of curcumin has been addressed, another bottleneck in the

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oral delivery of curcumin is its aqueous stability. The aqueous stability of soluble CEMCs was

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assessed for 24 h at pH 1.2, 4.5, 6.5, or 7.4 with curcumin or the equivalent amount of CEMCs

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for 24 h. Curcumin or the curcumin equivalent of CEMCs were dissolved in respective buffers containing 10 % methanol and incubated at 37 ˚C, 10% methanol was used to reach optimum

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solubility of curcumin at initial stages for the detection of curcumin even after 80 % of

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degradation. Addition of 10 % methanol did not alter the complex formation (data not shown). As the stability data indicates (Fig. 9), unformulated curcumin degrades very rapidly in aqueous

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buffers. The rate of degradation was more in solution with neutral and basic pH compared to

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solution with the acidic pH. In contrast, the degradation of curcumin was significantly reduced when complexed with Eudragit® EPO. However, the trend of its sensitivity to basic pHs

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continued even with CEMCs.

3.11 CEMCs Enhance the Oral Bioavailability of Curcumin in Mice. Finally, the oral bioavailability of curcumin in fasting BALB/c mice was investigated by delivering curcumin as unformulated curcumin or as CEMCs. The plasma concentration of curcumin was determined and plotted against time. The CEMCs achieved significantly (p <0.05) enhanced oral bioavailability compared to curcumin two hours after oral administration (Fig. 10). Pharmacokinetic parameters of the study (Table 3) shows that the CEMCs yielded a maximum plasma concentration (Cmax) of around 4.7 µg/ml, which is approximately 6 times higher than concentration achieved when fed with unformulated curcumin. The area under the concentration versus time curve (AUC) was also significantly improved (~ 20 times) when curcumin was delivered as CEMCs compared to unformulated curcumin. 17

ACCEPTED MANUSCRIPT Table 3 Plasma pharmacokinetic parameters Cmax, Tmax and AUC of curcumin and CEMCs after oral

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administration. Data represents mean ± standard deviation (n=3). * indicates variance were found

Curcumin

0.8±0.20

CEMCs

4.7±2.17*

AUC

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(µg/ml)

(h)

(µg*h/ml)

1.0±0.57

4.0±3.05

2.0±0

81.9 ±11.47*

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Group

Tmax

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Cmax

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Treatment

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significant (p< 0.05) using student’s t-test as compared to curcumin.

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4. Discussion

Curcumin is a plant derived, dietary supplement which is known to possess various

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pharmacological activities that are beneficial in multiple chronic diseases (Ghosh et al.). However, the concern of poor oral bioavailability posits significant pharmacokinetic barriers to translate its success to clinical application (Aggarwal et al., 2003; Anand et al., 2007; Gupta et al., 2013a; Gupta et al., 2013b). In this study, we have employed a completely novel and simple strategy of forming molecular complexes of curcumin with very safe polymer Eudragit® EPO (CEMCs). This formulation tremendously enhanced the aqueous solubility and the stability of curcumin and more importantly, increased the oral bioavailability of curcumin in mice by several folds. To translate the success of curcumin as a therapeutic agent from animal models to clinical applications, three challenges need to be addressed: poor aqueous solubility, poor aqueous stability, and high metabolism in the intestine and liver. To address these formulation issues,

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ACCEPTED MANUSCRIPT several approaches have been explored so far as mentioned under the introduction of this manuscript and reviewed thoroughly in the existing literature (Anand et al., 2007; Bansal et al.,

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2011; Mahmood et al., 2015; Siviero et al., 2015). However, majority of these approaches are not

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suitable for commercial use of curcumin as a daily oral supplement because of the high cost of these polymers (liposomes, PLGA, etc.) and the complexity of the preparation.

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Recently, solid dispersions using various hydrophilic polymers (PVP K-30, PEG 4000,

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PEG 6000, and Polyacrylates etc.) have started gaining attention as cost-effective alternatives in enhancing the solubility of various water insoluble drugs including curcumin (Chuah et al., 2014;

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Wegiel et al., 2014). In 2015, several reports suggests that solid dispersions of curcumin with Eudragit® EPO could enhance the solubility of curcumin, and improve its dissolution profile

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(Gangurde et al., 2015; Kerdsakundee et al., 2015; Li et al., 2015; Meng et al., 2015). However, Curcumin-Eudragit® EPO solid dispersions are yet to be tested for their ability to enhance in-

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vivo bioavailability. This manuscript reports that Eudragit® EPO forms molecular complexes with curcumin (CEMCs), which are different from solid dispersions in multiple aspects and importantly, CEMCs enhanced the in-vivo bioavailability of curcumin in mice. CEMCs were amorphous in nature, similar to previously reported solid dispersions, as shown by DSC, XRD and SEM. Interestingly, the interactions between curcumin and Eudragit® EPO remain intact even after dissolution into aqueous liquids (Fig.1) suggesting a complex formation between curcumin and the polymer. Polymers can interact with drug molecules by hydrogen bonds, ionic interactions, and/or Van der Waals forces to form drug polymer complexes (Nie et al., 2015). In solid state, as shown in FTIR spectrum (Fig. 2), the -OH band of curcumin was completely diminished in CEMCs. The disappearance of these peaks might be due to the interaction between the –OH group of

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ACCEPTED MANUSCRIPT curcumin and dimethylamino group of Eudragit® EPO as predicted by previous studies (Kerdsakundee et al., 2015; Li et al. 2015; Meng et al., 2015). Similar polar interaction between

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Eudragit® EPO and other drugs such as indomethacin and mefenamic acid have been reported,

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where such ionic interactions were shown to enhance the stability of the system (Chokshi et al., 2008; Kojima et al., 2012; Liu et al., 2010; Nie et al., 2015).

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The H-bonding interactions described in solid state of CEMCs (Fig. 2) may not exist in

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solution state in aqueous buffers. However, in aqueous solutions, curcumin still existed in complexation with Eudragit® EPO (Fig. 1). Therefore, H-bonding may not be the main reason

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for the existence of CEMCs. Observations from NMR studies indicated that the interaction that existed in aqueous buffers was perturbed by the addition of organic solvents (DMSO) (Fig. 3) ). Similarly in methanol, the CEMCs separate into

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(Catenacci et al. 2014; Ding et al. 2014

curcumin and Eudragit® EPO as shown by the method described in Fig. 1 (data not shown).

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Therefore, the authors hypothesize that hydrophobic interactions between these molecules is critical for the formation of the complex and play an important role to stabilize the supersaturated solution of curcumin (Kojima et al., 2012). Molecular complexation might have also contributed to improved protection of the curcumin from aqueous degradation at various pHs relevant to gastrointestinal tract. Therefore, curcumin interacts with Eudragit ® EPO through both H-bonding and hydrophobic interactions depending on the state. Both ionic and non-ionic interaction could play an important role for improving the solubility, stability and dissolution behavior of poorly soluble drugs (Sarode et al., 2013). Although, we do not rule out the formation of complexes in previously reported curcumin solid dispersions, existence of molecular complexes that are stable in solution state is not obvious from solid dispersions.

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ACCEPTED MANUSCRIPT The stability of amorphous form such as CEMCs depends on multiple factors (Chokshi et al., 2008; Hancock and Zografi, 1997; Meng et al., 2015). The critical factors include, the glass

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transition temperature (Tg), hygroscopicity, purity, storage conditions and interactions with the

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polymer. Chokshi et al. in their study have formulated and stabilized amorphous formulation of low Tg drug indomethacin with selected polymers, Eudragit® EPO, PVP-VA, PVP-K30 to study

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the solubility and dissolution behavior. The study concluded that Tg alone cannot be the

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determining factor for improving the stability and solubility of the system but also the nature and concentrations of polymers also play a vital role in stabilizing the amorphous nature of the drug.

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By forming interactions between curcumin and Eudragit® EPO at molecular level, the solid state stability of the CEMCs is predicted to be improved as suggested by previous study with multiple

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polymers including Eudragit® EPO (Meng et al., 2015). In 2015, Li et al. have formulated curcumin-Eudragit® EPO solid dispersions that

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enhanced the aqueous stability of curcumin (Li et al., 2015) as we have shown in this manuscript (Fig. 9). However, the Li et al. did not perform solubility and oral bioavailability studies on these dispersions. The results of Li et al. further confirms the observations of our study. Interestingly, Kerdsakundee et al. developed novel raft forming systems incorporating curcumin-Eudragit® EPO solid dispersions to prolong the gastric residence time and provide controlled local delivery of curcumin for gastric ulcers (Kerdsakundee et al., 2015). However, the technology presented in the current piece of work is aimed to enhance the oral bioavailability of curcumin for systemic purposes. CEMCs enhanced apparent solubility of curcumin by more than 20,000 times. CEMCs have achieved an AUC ~20-fold higher and a peak plasma concentration 6-fold higher than curcumin, which is still significant enough for clinical applications, however, not to an extent predicted by fold enhancement in aqueous solubility. This discrepancy is very well explained by

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ACCEPTED MANUSCRIPT Frank et al. through solid dispersions of poorly soluble drug ABT-102 (Frank et al., 2012). Their results suggest that permeation rate enhancement is mainly due to the availability of molecularly

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dissolved drug rather than apparent solubility in presence of surfactants.

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CEMCs, with improved bioavailability, will have significant applications in the prevention and treatment of several complex chronic diseases such as cancer, atherosclerosis,

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diabetes, Alzheimer’s disease, inflammatory bowel syndrome, arthritis, cystic fibrosis, malaria

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etc. that require daily medication. For daily medication/supplement for chronic therapies, oral route is preferred. CEMCs are expected to overcome bioavailability related roadblocks to

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translate curcumin as a potential drug or supplement for prevention/treatment of these chronic diseases. More studies are however needed to study the efficacy of CEMCs in animal models of

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several chronic diseases as well as clinical models in humans. The currently formulated curcumin-Eudragit® EPO molecular complexes has several

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competitive advantages over existing technologies to increase the bioavailability of curcumin: high aqueous solubility and loading, stable at wide range of pHs, cost effective, easy procedure to scale up, and use of FDA approved ingredients. This is a proof of concept study that has great potential to enhance the therapeutic efficiency of curcumin in various complex chronic diseases. 5. Conclusion

The present study is based on the preparation of a novel formulation that potentially enhances the oral bioavailability of curcumin by increasing its aqueous solubility and stability. Curcumin formed molecular complexes with Eudragit® EPO that are stable in solution state. This complexation enhanced the aqueous solubility of curcumin from ~1 µg/ml to >20 mg/ml. In addition to the stabilization of supersaturated solution of curcumin, the complexation also enhanced its aqueous stability at various pHs. Finally, formulating curcumin through CEMCs 22

ACCEPTED MANUSCRIPT enhanced the oral bioavailability of curcumin in mice by several folds (AUC; ~20 times and Cmax; ~6 times).

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Author Information

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* Corresponding author: Associate Professor, Department of Pharmaceutical Sciences, College

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of Pharmacy, South Dakota State University, SAV # 255, Box 2202C Brookings, South Dakota

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57007, United States. E-mail: [email protected]. Notes

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The authors declare the following conflicts of interest. Drs. Sunny Kumar and corresponding

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author Hemachand Tummala are authors on the patent application filed for this technology.

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Acknowledgements

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Authors are thankful to the Department of Pharmaceutical Sciences, South Dakota State University (SDSU) and the research support funds from the office of research, SDSU for funding the study.

We acknowledge technology transfer office (TTO), SDSU for filing a patent

application. We are grateful to Evonik Industries for providing us free sample of Eudragits®. Abbreviations

GRAS- Generally Recognized as Safe FDA- United States Food and Drug Administration PVA- Polyvinyl Alcohol CEMCs-Curcumin- Eudragit® EPO molecular complexes FTIR- Fourier Transform Infrared FTIR Spectroscopy

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ACCEPTED MANUSCRIPT DSC- Differential scanning Calorimetry

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XRD- X-ray Diffraction SEM- Scanning Electron Microscopy

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HPLC- High-Performance Liquid Chromatography

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ACCEPTED MANUSCRIPT Table Captions

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Table 1 Optimization of loading and apparent solubility (in 4 hrs) of curcumin from CEMCs prepared

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using three different organic solvents and stabilizer. Data represents mean ± standard deviation

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(n=3). * indicates variance is significant (p <0.05) as compared to sample 1 and 3. ǂ indicates

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variance is significant (p <0.05) as compared to sample 4. Table 2

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Effect of curcumin to polymer ratio on the loading of curcumin in CEMCs prepared using

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acetone as a solvent and PVA as stabilizer. Data represents mean±standard deviation (n=3)

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Table 3

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Plasma pharmacokinetic parameters Cmax, Tmax and AUC of curcumin and CEMCs after oral administration. Data represents mean ± standard deviation (n=3). * indicates variance were found significant (p< 0.05) using student’s t-test as compared to curcumin.

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Figure Legends

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Fig. 1. Curcumin–Eudragit® EPO molecular complex (CEMC) formation: Curcumin and

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curcumin from CEMCs were dissolved in the buffers as shown in the figure. The ratio of the

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concentration of soluble curcumin present in solution after passing through the 10 kDa cut-off membrane to the concentration measured before filtration were compared. Complex formation

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with high M.Wt-Eudragit® EPO will prevent curcumin to filter through the membrane.

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Fig. 2. FTIR spectrum of Curcumin (in red), Eudragit® EPO polymer (in purple), physical mixture of curcumin and Eudragit® EPO (in Green) and CEMCs (in Cyan). As can be seen in

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CEMCs (in Cyan) the -OH band of curcumin was completely diminished in CEMCs.

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Fig. 3. NMR Spectrum of CEMCs in the following solvents. (A) 1:1 D2O: acidic H2O. (b) 1:1

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acidic H2O DMSO-d6. (C) 1:3 acidic H2O DMSO-d6. Fig. 4. Differential scanning calorimeter (DSC) endothermic curves of (A) Curcumin, (B) Eudragit® EPO polymer, (C) the physical mixture of curcumin and Eudragit® EPO, and (D) CEMCs. X-axis represents temperature in º C whereas Y-axis represents the heat flow in (W/g). Fig. 5. X-ray diffraction patterns (XRD) curves of (A) Curcumin, (B) Eudragit® EPO polymer, (C) physical mixture of curcumin and Eudragit® EPO, and (D) CEMCs. X-axis represents temperature from 0°C to 60°C Fig. 6. Scanning electron microscopy (SEM) images of CEMCs at an accelerating voltage of 10 kV. The dry samples were mounted on metal holders using conductive double-sided tape and sputter coated with a gold layer for analysis.

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ACCEPTED MANUSCRIPT Fig. 7. Apparent solubility of curcumin in pH 1.2 (0.1N HCl) at 37˚C 4 h with 100 rpm shaking. Curcumin, the physical mixture of curcumin and Eudragit® EPO, and CEMCs were dispersed in

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1ml of pH 1.2 (0.1N HCl). The curcumin dissolved after 4 h was analyzed by UV absorbance at

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420 nm. CEMCs resulted in significantly high apparent curcumin solubility than unformulated curcumin. Data represents mean ± standard deviation (n=3). * indicates variance were found

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significant (p< 0.05) using student t-test as compared to curcumin.

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Fig. 8. Solubility of CEMCs at different pHs in comparison to curcumin Panels A and B

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represents soluble curcumin after dissolving the equivalent quantity of curcumin (5 mg/ml) from CEMCs or curcumin, respectively in buffers with various pHs (pH 1.2, pH 4.5 and pH 7.4). The

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Fig. 9. The aqueous stability of soluble CEMCs was assessed at pH 1.2, 4.5, 6.5, or 7.4 with

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curcumin or the equivalent amount of CEMCs. Fig. 10. Curcumin oral bioavailability. Curcumin (150 mg/kg) or equivalent CEMCs were given to mice orally. Blood samples were collected from the mice at different time points (0.5, 1, 2, 4, 8, 12, and 24 h). Curcumin was extracted from the plasma and analyzed via reverse HPLC with UV absorption at 420 nm. Data represents mean ± standard deviation (n=3).

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Graphical abstract

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