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Accepted Manuscript
Site-directed polymer-drug complexes for inflammatory bowel
diseases (IBD): Formulation development, characterization, and
pharmacological evaluation
Siddharth S. Kesharwani, Rizwan Ahmad, Mohammed Ali
Bakkari, Mrigendra K.S. Rajput, Rakesh Dachineni, Chaitanya
K. Valiveti, Saurabh Kapur, G. Jayarama Bhat, Amar B. Singh,
Hemachand Tummala
PII:
DOI:
Reference:
S0168-3659(18)30457-7
doi:10.1016/j.jconrel.2018.08.004
COREL 9413
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
17 March 2018
20 July 2018
2 August 2018
Please cite this article as: Siddharth S. Kesharwani, Rizwan Ahmad, Mohammed Ali
Bakkari, Mrigendra K.S. Rajput, Rakesh Dachineni, Chaitanya K. Valiveti, Saurabh
Kapur, G. Jayarama Bhat, Amar B. Singh, Hemachand Tummala , Site-directed polymerdrug complexes for inflammatory bowel diseases (IBD): Formulation development,
characterization, and pharmacological evaluation. Corel (2018), doi:10.1016/
j.jconrel.2018.08.004
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Site-Directed Polymer-Drug Complexes for Inflammatory Bowel Diseases (IBD): Formulation
Development, Characterization, and Pharmacological Evaluation
Siddharth S. Kesharwania, Rizwan Ahmadb, Mohammed Ali Bakkaria,c, Mrigendra K.S. Rajputa,
Rakesh Dachinenia, Chaitanya K. Valivetia, Saurabh Kapurb, G. Jayarama Bhata, Amar B.
Department of Pharmaceutical Sciences, College of Pharmacy & Allied Health Professions,
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a
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Singhb* and Hemachand Tummalaa*
South Dakota State University, Brookings, SD-57007, USA.
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center,
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College of Pharmacy, Jazan University, Jazan-45142, SA.
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c
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Omaha, NE-68198, USA.
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* Corresponding author
Associate Professor
* Co-corresponding author
Associate Professor
Department of Biochemistry and Molecular
College of Pharmacy & Allied Health Professions
Biology,
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Department of Pharmaceutical Sciences
South Dakota State University
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SAV # 255, Box 2202C
University of Nebraska Medical Center,
Omaha, NE-68198, USA
Brookings, SD, 57007, USA
Phone no: +1-402-559-6340
Phone no. +1-605-688-4236
E-mail: amar.singh@unmc.edu
Fax +1-605-688-5993
E-mail: hemachand.tummala@sdstate.edu
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ABSTRACT
Inflammatory Bowel Diseases (IBD) is a debilitating condition that affects ~70,000 new
people every year, and has been described as “an expensive disease with no known cure”. In
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addition, IBD increases the risk of developing colon cancer. The current therapeutics for IBD
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focus on the established disease where the immune dysfunction and bowel damage have
already occurred but do not prevent or delay the progression. The current work describes a
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polymer-based anti-inflammatory technology (Ora-Curcumin-S) specifically targeted to the
luminal side of the colon for preventing and/or treating IBD. Ora-Curcumin-S was prepared
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by molecular complexation of curcumin with a hydrophilic polymer Eudragit® S100 using
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co-precipitation method. Curcumin interacted with the polymer non-covalently and existed in
an amorphous state as demonstrated by various physicochemical techniques. Ora-Curcumin-
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S is a polymer-drug complex, which is different than solid dispersions in that the interactions
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are retained even after dissolving in aqueous buffers. Ora-Curcumin-S was >1000 times
water soluble than curcumin and importantly, the enhanced solubility was pH-dependent,
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which was observed only at pHs above 6.8. In addition, around 90% of Ora-Curcumin-S was
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stable in phosphate buffer, pH 7.4 and simulated intestinal fluid after 24 h, in contrast to 1020% unformulated curcumin. Ora-Curcumin-S inhibited Monophosphoryl Lipid-A and E.coli
induced inflammatory responses in dendritic cells and cells over expressing Toll-Like
Receptor-4 (TLR-4) suggesting that Ora-Curcumin-S is a novel polymer-based TLR-4
antagonist. Preliminary pharmacokinetics in mice showed targeted delivery of soluble
curcumin to the colon lumen without exposing to the systemic circulation. Furthermore, OraCurcumin-S significantly prevented colitis and associated injury in a mouse model of
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ulcerative colitis estimated using multiple preclinical parameters: colonoscopy pictures, body
weight, colon length, colon edema, spleen weight, pro-inflammatory signaling and
independent pathological scoring. Overall, the outcome of this innovative proof-of-concept
study provides an exciting and locally-targeted pathway for a dietary therapeutic option for
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IBD patients to help limit colonic inflammation and thus susceptibility to colitis- associated
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KEYWORDS:
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colorectal cancer.
Polymer-drug complexes, Solubility & Stability, Site specific delivery,
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Inflammatory bowel disease, Toll-Like Receptor-4, Curcumin.
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1. INTRODUCTION
Inflammatory Bowel Diseases (IBD) is a conglomerate of auto-immune disorders characterized
by chronic inflammation, mucosal ulceration, edema and hemorrhage of the gastrointestinal (GI)
tract. In addition, IBD patients possess the considerable risk of developing colon cancer [1-3].
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The overall incidence of IBD, especially in the young adults, and the disease severity are
constantly increasing [4-7]. Thus, if safe preventive measures are not taken, the increased
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incidence of IBD and associated cancer in young adults will reach epic proportions by the time
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they reach old age [7]. Consequently, the existing anti-IBD therapies primarily comprising
immune-suppressive agents with severe side-effects could be life-threatening [8-10]. Moreover,
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current therapeutic interventions in IBD focus on an established disease where the immune
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dysfunction, dysbiosis, and bowel damage have already occurred. However, to truly alter the
clinical management of this disease, intervention should ideally start at an early stage or before
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potential remission (in a patient with active disease). Such an approach can not only produce a
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better outcome and lower levels of cancer susceptibility but will also sharply decrease
hospitalization rate and surgeries.
However, discovering novel drugs is an expensive and
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extremely time-consuming process [10]. Therefore, a pragmatic approach to address this gap for
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improving the clinical management of the disease could be to repurpose the existing and
validated safe anti-inflammatory compounds by circumventing their translational roadblocks
using unique delivery strategies.
In this regard, curcumin, a well-established safe anti-inflammatory compound [11, 12],
has been recently identified to alter the function of Toll-like receptors (TLRs) [13, 14]. Although
etiology of the IBD is still evolving, several recent studies using genetic, physiological and
pharmacological approaches have provided evidence that the deregulation of TLR-4 and
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associated signaling promotes IBD severity and susceptibility to colitis-associated cancer (CAC)
[15-17]. Notably, in the pathological communication between the luminal antigens and the
mucosal immune components, a critical role of Toll-like Receptors (TLRs)-MyD88 signaling is
well recognized [18]. One of the essential roles played by TLRs has been their ability in
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recognizing structurally conserved molecules derived from the microbes and thus play an
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essential role in the innate immune system and inflammatory signaling. In this regard, In
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accordance, an increased TLR-4 expression is reported in IBD patients compared to the healthy
humans [19, 20]. Physiologically, altered TLR-4 signaling has been linked to the modulation of
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mucosal barrier properties [19, 21, 22], intestinal microbiota [23], and immune activation; three
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changes that are connected to IBD pathobiology and/or associated colorectal cancer. Of interest,
considering its essential role in mucosal immune homeostasis and causal association with IBD
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and associated cancer, TLR-4 has been identified as a highly effective immune therapeutic target
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to minimize IBD-associated morbidity and subsequent susceptibility to the colon cancer.
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Curcumin is well tolerated in humans even at very high doses (up to 12 g/day) [24] and
therefore recognized as safe (GRAS) by the United States Food and Drug Administration (FDA).
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Furthermore, several pre-clinical studies have provided strong evidence for the use of curcumin
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in ameliorating inflammatory diseases, including IBD [11, 12]. However, despite the known
medicinal value, its clinical application is yet to be successful, primarily due to its failure to
consistently maintain the required therapeutic blood levels [24-28]. Extremely poor water
solubility (<1 µg/ml) of curcumin, that leads to impaired oral absorption, along with its high
metabolic rate contributes to its poor bioavailability. Therefore, recent focus has been to improve
the bioavailability of curcumin with new fomulations, which resulted in an overall increase in its
blood levels but not consistently long enough for the clinical advancement [29-34]. Despite
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achieving higher blood levels for curcumin, one overlooked factor is that such high systemic
levels could cause metabolic interactions with others drugs through its inhibition of cytochromeP450 (CYP-450) enzymes and efflux transporters such as P-glycoprotein (P-gp) [35-40].
In contrast to the majority of the previously reported strategies, current work describes a
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highly water soluble polymer-drug complex (hereon Ora-Curcumin-S) of curcumin with a
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hydrophilic pH-dependent polymer Eudragit® S100 to specifically deliver anti-inflammatory
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curcumin to the luminal side of the colon without significantly exposing to the blood circulation
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(minimize systemic absorption). Ora-Curcumin is reported as a new polymer-based TLR4
antagonist as shown by using multiple immune cells (dendritic and genetically modified kidney
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epithelial cells). The complexation significantly improved the pharmacokinetic concerns by
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significantly enhancing the aqueous solubility and stability of the curcumin, and also imparted an
ability to specifically target curcumin locally to the target colon tissue at the luminal side.
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Moreover, we have demonstrated that Ora-Curcumin-S is highly effective in ameliorating colitis
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using in-vivo mouse model, potentially by interfering with TLR4 signaling. Overall, we provide
a design of a polymer-curcumin based TLR4 antagonist and a strong preclinical evidence
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supporting the local delivery and efficacy of the long-sought clinical application of curcumin for
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improving IBD pathobiology and the associated risk of colon cancer.
2. MATERIALS & METHODS
Materials and Reagents
The Eudragit® S100 polymer was provided by Evonik Industries (Birmingham, AL, USA).
Curcumin (TCI America) (> 98% pure), solvents of HPLC grade (acetone, dimethyl sulfoxide
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(DMSO), methanol, ethanol); stabilizers/surfactants were purchased from Fisher Scientific
(Pittsburgh, PA, USA). Surfactants Tween-20 and Pluronic F-68 were purchased from Amresco
(Solon, OH, USA), and Panreac AppliChem (St. Louis, MO, USA) respectively. Tumor necrosis
factor-α (TNF-α) and Interleukin-8 (IL8) ELISA kits and Monophosphoryl Lipid A (MPLA)
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were purchased from e-Biosciences, Inc. and InvivoGen, (San Diego, CA, USA) respectively.
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Dextran Sodium Sulfate (DSS) was purchased from TdB Consultancy (Uppsala, Sweden). Anti-
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NF-kB antibody was purchased from Cell Signaling Technology (Danver, MA, USA) while
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Periodic Acid-Schiff (PAS) Kit from Sigma (St. Louis, MO, USA). Vectastain ABC and DAB
kits for immunostaining were purchased from Vitor Labotaries (Burlingame, CA, USA). RNA
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isolation Kit was purchased from Qiagen (Valencia, Santa Clarita, CA, USA) while quantitative
real-time PCR reagents were procured from Bio-Rad (Hercules, California, USA). Real-time
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PCR primers were synthesized by integrated DNA Technology, Inc. (Skokie, Illinois, USA).
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Cell Lines and Cell Culture
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Human origin HEK-TLR-4YFP-MD-2 cell line (NR-9315) was obtained through the BEI
resources, ATCC (Manassas, VA, USA). HCT116 (Human colorectal carcinoma) and HT-29
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(Human colorectal adenocarcinoma) were purchased from the ATCC (Manassas, VA, USA). We
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cultured the cell lines in DMEM-high glucose medium in a humidified atmosphere of 5% CO2 at
37ºC. Dendritic cells (DC2.4) cells were cultured in DMEM-high glucose medium in presence of
50 µM of ß-mercaptoethanol. The culture media also contain 10% fetal bovine serum (FBS) and
penicillin/streptomycin.
Preparation of Curcumin-Eudragit® S100 Polymer Complexes (Ora-Curcumin-S)
Ora-Curcumin-S was prepared by co-precipitating the drug and the polymer as described
previously with slight modifications [41]. The drug (Curcumin) and the polymer (Eudragit®
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S100) were dissolved in an organic solvent (4 ml). The rationale for the selection of organic
solvents is based on their ability to dissolve both the curcumin and the Eudragit® S100 and their
dielectric constant. Subsequently, to achieve controlled precipitation, the solution containing
curcumin-Eudragit® S100 was added into an acidic aqueous solution containing 3% w/v of a
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surfactant/stabilizer under constant stirring (400 RPM). The evaporation of the organic solvent
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was achieved by stirring the dispersion (~16 h). Following completion of 16 h, the dispersion
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was centrifuged at 20,000 g for 30 min to collect the precipitated complexes. The complexes
were resuspended in deionized water, freeze-dried and stored protected from light in dark at < -
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20˚C until further use. Since curcumin in unstable/degrades in presence of light, we performed
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all the procedures in the dark.
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Formulation Optimization of Ora-Curcumin-S
The optimization of Ora-Curcumin-S preparation was achieved by changing various formulation
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parameters such as the type of organic solvent, type of surfactant/stabilizer and the curcumin to
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Eudragit® S100 polymer ratio. The types of an organic solvent selected were acetone, ethanol,
methanol, and acetone+DMSO). The type of surfactant/stabilizer modified included polyvinyl
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alcohol (PVA), Pluronic F-68 and Tween-20. The drug to polymer ratio altered included (1:2,
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1:5, 1:10 and 1:20) to further enhance the curcumin loading without compromising curcumin
solubility [41].
Assessment of Polymer-Drug Complex Formation
The polymer-drug complex formation was assessed by dissolving or dispersing curcumin and
Ora-Curcumin-S in absolute ethanol or 50 mM phosphate buffer pH 7.0 containing 0% or 10%
ethanol. The samples were subjected to centrifugation at 20,000 g for 20 min. The supernatant
was collected and subsequently passed through Spin-X® UF concentrator tubes (10 kDa cutoff
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membrane). The ration (bottom/top) was calculated as the concentration of curcumin in the
solution before (top of the tube) and after passing through the 10kDa cut-off membrane (bottom
of the tube) was determined at 420 nm using a UV−Visible spectrophotometer [41].
Fourier Transform Infrared (FTIR) Spectroscopy
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The complex formation was also confirmed by recording the FTIR spectrum on Nicolet 380
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ATR-FTIR spectrophotometer (Thermo Electron Corp., Madison, WI). Curcumin, Eudragit®
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S100, curcumin and Eudragit® S100 physical mixture, and Ora-Curcumin-S spectrum were
acquired between 4000 cm−1 and 400 cm−1 at a scanning speed of 4 cm−1. The data represents the
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average of 50 scans of the data [42, 43].
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Nuclear Magnetic Resonance (NMR) Spectroscopy
To estimate the nature of interaction driving the formation of Ora-Curcumin-S, 1H-NMR spectra
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of Ora-Curcumin-S were recorded on a Bruker 400 MHz NMR spectrometer. Briefly, 30 mg of
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Ora-Curcumin-S was dissolved in 1 ml of alkaline D2O (0.25N Na2CO3), 100 % of DMSO-d6 or
50 % DMSO-d6 in alkaline D2O. The synthetic curcumin and the polymer Eudragit® S100 were
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dissolved in DMSO-d6. The NMR spectra were recorded for all the solutions [42, 43].
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Scanning Electron Microscopy (SEM)
The surface morphology of Ora-Curcumin-S was investigated using scanning electron
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microscopy (SEM FEI, Quanta 450 SEM, GEAR Laboratory, USD Biomedical Engineering).
The freeze dried Ora-Curcumin-S complex was sputter coated with a 10-nm gold layer by
mounting on metal holders using conductive double-side tapes. The images were captured at a
various accelerating voltage of 5-20 kV. [41].
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Differential Scanning Calorimetric (DSC) and Powder X-ray Diffraction (XRD) Analysis
To determine the solid state nature of the complexes, we performed differential scanning
calorimetry (DSC) and powder X-ray diffraction (XRD) analysis respectively. Curcumin,
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Eudragit® S100, curcumin and the Eudragit® S100 physical mixture and Ora-Curcumin-S were
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analyzed. DSC analysis was performed using TA Instruments Q200 Differential Scanning
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Calorimeter (TA Instruments, New Castle, DE, USA) and XRD was done using Rigaku powder
X-ray diffractometer with Cu radiation, running at 40 kV and 44 mA. The procedure for both the
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analysis was similar to our previously reported studies and is described in sufficient detail in the
Curcumin Loading in Ora-Curcumin-S
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following references [41, 43].
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Curcumin loading (µg of curcumin present per mg of total Ora-Curcumin-S) was evaluated by
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the following procedure. Briefly, 5 mg of Ora-Curcumin-S was dissolved in 1 ml of dimethyl
sulfoxide (DMSO) and the curcumin was extracted. The amount of curcumin present was
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measured at 420 nm using a UV−Visible spectrophotometer. A standard curve prepared by using
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synthetic curcumin in DMSO was used to calculate the concentration of the curcumin in the
extract. The interference of the other formulation components at 420 nm was ruled out before
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establishing the method for analysis [41].
Apparent Solubility of Ora-Curcumin-S
The apparent solubility of Ora-Curcumin-S was evaluated by following the same procedure as
previously reported with slight modifications. Apparent solubility could be defined as the
solubility that is measured after 4 h and not the equilibrium solubility. In brief, curcumin or
curcumin equivalent of Ora-Curcumin-S were dispersed in pH 7.0 solution and incubated at 37
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°C for 4 h with 100 RPM shaking. After 4 h, the samples were subjected to centrifugation at
20,000 g for 20 min. The supernatant was collected and filtered through 0.2 µM filter. The
curcumin concentration was calculated at 420 nm by using a UV-visible spectrophotometer [41].
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Aqueous Stability of Ora-Curcumin-S
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Ora-Curcumin-S aqueous stability was measured using simulated intestinal fluid TS (SIF) (pH
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6.7-6.9) and 50 mM phosphate buffered saline (PBS), pH 7.0 buffer. Briefly, 5 mg of curcumin
or the curcumin equivalent of Ora-Curcumin-S were solubilized in SIF and pH 7.0 buffer. Ten
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percent methanol was added to both SIF and pH 7.0 buffer to solubilize the synthetic curcumin
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and to enable detection even after degradation of curcumin and incubated at 37 ˚C, 100 RPM
shaking. At predetermined time intervals (0-16 h); samples were collected and processed as
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mentioned for aqueous solubility. The amount of curcumin in the filtrate was determined by
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using a UV−Visible spectrophotometer at 420 nm provided us the information about soluble and
stable curcumin [41].
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pH-dependent Solubility of Ora-Curcumin-S
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The pH-dependent solubility of unformulated curcumin and curcumin equivalent of OraCurcumin-S was evaluated in various buffers with pHs 1.2, 4.5, 7.0 and 7.4. The qualitative
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solubility pattern of curcumin and Ora-Curcumin-S in the above mentioned buffers is depicted as
a photograph [41].
pH-dependent Drug Release Kinetics of Ora-Curcumin-S
The in-vitro release of curcumin and curcumin from Ora-Curcumin-S was evaluated in a buffer
with gradually changing pHs (pH 1.2, 4.6, 6.7 and 7.4). The buffer solutions were selected to
mimic the normal pH variations encountered along the entire length of the gastrointestinal tract
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(GIT). Samples were collected at different time points (0-16 h), subjected to centrifugation at
20,000g for 20 min, the supernatant collected and filtered through 0.2 μM filter. The amount of
curcumin was determined by using a UV−Visible spectrophotometer at 420 nm [44].
pH-dependent Intracellular Delivery of Curcumin by Ora-Curcumin-S
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The pH-dependent cellular uptake of curcumin and Ora-Curcumin-S were determined on
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HCT116 and HT29 colon cancer cells by using flow cytometry. Briefly, 1×105 cells/well were
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cultured in 24-well plates and incubated with the synthetic curcumin and Ora-Curcumin-S in 50
mM citrate buffer, pH5.5, and 50 mM phosphate buffer, pH 7.0. After 4 h at 37 ˚C, the cells were
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washed with PBS, trypsinized, fixed using 4% paraformaldehyde (PFA) and stored at 4°C until
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further analysis. The amount of curcumin or Ora-Curcumin-S associated per cell was quantified
using flow cytometer equipped with a blue laser (488 nm). Sample acquisition was performed
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using BD Biosciences FACS LSR Fortessa and data analyzed using FlowJo software [43].
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Systemic Exposure and Colon-Targeting Ability of Curcumin Formulations in Mice
All animal experiments described here were performed in compliance with regulations of the
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Institutional Animal Care and Use Committees (IACUC) of South Dakota State University,
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Brookings, SD, USA. BALB/cJ mice (n = 5 per group, 6 − 8 weeks old) purchased from Jackson
laboratories (Maine, USA) were used for drug distribution studies. Curcumin (15 mg/kg),
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curcumin equivalent of Ora-Curcumin-S or Ora-Curcumin-E (previously reported curcumin
formulation with Eudragit® EPO) [41] were orally administered to fasting mice in 10 mM citrate
buffer (pH 4.5) as a suspension or solution. Blood samples and fecal matter from the mice were
collected 1 h and 16 h after administering the formulations, respectively. One hour time point is
chosen for blood collection based on our previous results as the Tmax for curcumin in mice [41].
Acetonitrile was used to extract the curcumin from the mouse plasma immediately. Reverse
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phase high-performance liquid chromatography (HPLC) equipped with a UV-detector was
employed for detection of the amount of curcumin in mouse plasma (See method below for
HPLC).
Estimation of Soluble and Insoluble Curcumin in Fecal Samples
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Fresh fecal sample from each mouse was collected by compressing the abdomen gently and
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immediately snap-frozen using liquid nitrogen. The samples were stored at -80 °C in the dark
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until further analysis. The soluble and insoluble fractions of curcumin were isolated from the
fecal pellets after weighing each pellet. The fecal pellets were crushed and 500 μl of pH 7.0
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phosphate buffer was added, sonicated for 60 seconds, centrifuged at 20,000g for 10 min to
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obtain water soluble curcumin in the supernatant and water insoluble curcumin in the pellet. The
supernatant was filtered through 0.2 m filter. To 300 μl of the supernatant collected (soluble
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fraction), a 700 μl of acetonitrile was added before analyzing with HPLC. To the insoluble pellet,
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a 700 μl of acetonitrile was added directly, vortexed for 3-5 min, and centrifuged to collect the
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water-insoluble fraction of curcumin in the acetonitrile. A correction factor was added to the
calculations of insoluble fractions to accommodate the soluble curcumin in the pellet with the
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unresolvable phosphate buffer (~200 l). The amount of curcumin present in both the fractions
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was analyzed using HPLC as described below after passing the solutions through 0.2 m filter.
Quantification of Amount of Curcumin by Reverse Phase HPLC
Chromatographic separation of curcumin was achieved by using Symmetry® C18 column (150
mm x 4.6 mm, 5 µm, Waters, USA). An isocratic elution using acetonitrile and 1% w/v citric
acid buffer (pH 3.0) (60:40 v/v ratio) at a flow-rate of 1 ml/min was used for detection.
Curcumin was detected using a UV detector at 420 nm [41, 45].
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Activation of Dendritic Cells (DC2.4): MPLA or E. coli Induced Cytokine Release
DC2.4 cells (5.0 x 105) were treated with either solubilized curcumin (using DMSO) or curcumin
equivalent of Ora-Curcumin-S (dose, 5 and 10 µg/ml) for 1 h. Subsequently, cells were
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stimulated with MPLA (2 µg/ml) or E. coli (5.0 x 105 /ml). After 48 h of stimulation, the
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concentration of TNF- was measured using TNF- ELISA kit from e-Biosciences Inc. as an
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activation marker [42, 43].
TLR-4 Inhibition Assay: Dose-Response Curve
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The dose-response curve of curcumin or Ora-Curcumin-S on inhibiting the MPLA-induced TLR-
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4 activation was tested using HEK293TLR4YFPMD2 cells (5.0 x 105 cells/well) by pretreating the
cells with curcumin or curcumin equivalent of Ora-Curcumin-S (1-50 µg/ml) for 1 h.
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Subsequently, cells were challenged with MPLA (2 µg/ml) to activate TLR-4. After 48 h of
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activation, the concentration of IL-8 was estimated by performing ELISA using IL-8 kit from ebiosciences as an indicator of TLR-4 activation.
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In another set of experiments, the efficacy of curcumin or equivalent of Ora-Curcumin-S
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in antagonizing the activation of TLR-4 by increasing concentrations of MPLA was assessed
using HEK293TLR4YFPMD2 cells. A total of 5.0 x 105 cells were pre-treated with the curcumin
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or curcumin equivalent of Ora-Curcumin-S (5 µg/ml) followed by the addition of MPLA (3-6
µg/ml). After 48 h of activation, as an indicator of TLR-4 activation, the concentration of IL-8
was estimated by performing ELISA using IL-8 kit from e-biosciences
Cell Viability Assay
The effect of curcumin and Ora-Curcumin-S on the viability of HCT116 and HT29 human colon
cancer cells was determined by the MTT assay method. Curcumin was dissolved either in
dimethyl sulfoxide (DMSO) or pH 7.0 buffer, and Ora-Curcumin-S was dissolved in pH 7.0
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buffer as a stock solution. Briefly, 50,000 cells/well were treated with varying (0-40 µM)
concentrations of curcumin and curcumin equivalent of Ora-Curcumin-S at 37°C. Following 48
h of incubation, 0.5 mg/ml MTT solution was added to each well, and the cells were incubated
for further 4 h at 37°C. Subsequently, after incubation for 4 h, 150 µl of DMSO was added to
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each well to dissolve the formazan crystals and further incubated for 1 h at 37°C. The absorbance
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was then recorded at 570 nm with a reference wavelength of 650 nm using a UV-visible
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spectrophotometer. The results are represented in terms of percentage inhibition of cell
proliferation compared to that of vehicle control. The cellular viability was assessed as a
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percentage of the control by using the following equation [46].
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Viability (%) =
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Therapeutic Potential of Ora-Curcumin-S on a Dextran Sodium Sulfate (DSS) Induced Colitis
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Mouse Model
C57BL/6J mice (8-10 weeks old) were used for the DSS induced colitis study. The mice were
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divided into three groups: healthy control, DSS, and DSS with Ora-Curcumin-S treatment. The
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mice were administered regular drinking water which was designated as the control group or
2.5% w/v DSS to induce colitis following the same procedure as previously reported [47, 48]. A
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15 mg/kg dose of (150 µl) of curcumin equivalent Ora-Curcumin-S was chosen for treatment in
this study. Each formulation was administered orally two days prior to the DSS administration
and subsequently every day during the entire experiment.
The mice were monitored for
DSS/water consumption. The body weight was recorded daily and the mice were monitored for
any signs of diarrhea and/or rectal bleeding. On day 5, two days prior to sacrificing, colonoscopy
was done to assess the extent of colonic inflammation. Mice were sacrificed at day 7, spleen and
colon tissue were harvested for further evaluation. To calculate the disease activity index (DAI),
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the combined scores of (a) weight loss, (b) stool consistency, and (c) bleeding were used. The
score was estimated as follows: change in weight (0: <1%, 1: 1–5%, 2: 5–10%, 4: >15%); stool
blood (0: negative, 2: positive or 4: gross bleeding; and stool consistency (0: normal, 2: loose
stools, 4: diarrhea), as previously described [48]. Body weight loss was calculated as the percent
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difference between the original body weight and the actual body weight on a given day while
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colon edema was calculated as the ratio of colon weight (gm) per colon length (cm). Colon Swiss
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role was made, stained with haematoxylin and eosin for histopathological examination. The
histological analysis of the sections was performed by a University of Nebraska Medical Center
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GI-pathologist in a blinded manner. The cumulative injury score was calculated according to
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supplemental Table 1.
Immunohistochemistry
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The procedure employed for immunohistochemistry involved deparafinization of tissue sections
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in xylene and subsequent rehydration in graded alcohols. Antigen retrieval was performed by
subjecting the tissue sections to 100°C for 20 min in Tris-EDTA buffer (pH 9.0). Endogenous
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peroxidase was blocked by adding H2O2 solution (3%) for 15 min at room temperature.
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Immunostaining was done according to manufacturer’s instructions (ABC kit victor Lab).
Briefly, for immunostaining, the sections were blocked in normal goat serum followed by
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incubation with primary antibody overnight. Tissue sections were incubated in presence of
biotinylated anti-goat for 30 min. Subsequently, tissue sections were incubated with Vectastain
ABC reagent for another 30 min. Then liquid Diaminobenzidine (DAB) was applied for 5 min.
counterstaining was achieved by using Mayer’s haematoxylin. Slides were washed in pH 7.4
PBS between each immunostaining step.
negative controls.
Appropriate controls were used as positive and
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Quantification of Pro-inflammatory and Anti-inflammatory cytokines by Real-Time PCR
To quantify the pro-inflammatory and anti-inflammatory cytokines in tissue samples, we
performed RNA isolation and subsequent qRT-PCR analysis. RNeasy kit was utilized for
extraction of RNA from tissue samples. iScript cDNA synthesis kit (Bio-Rad) was used for
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revere-transcribing 1 μg of RNA. Each PCR reaction was performed with 2 μl of cDNA and 2×
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iQTM SYBR Green Supermix (Bio-Rad). The primer sequences for TNF-α, IL-6, IL-10, Muc-2,
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and β-actin were described in the supplementary information (supplementary table-2) [48].
Periodic Acid Schiff (PAS) staining
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Periodic Acid Schiff-haematoxylin method was used to detect mucus secreting goblet cells.
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Briefly, the tissue sections on slides were dewaxed and hydrated. The PAS staining kit from
Sigma used and images were captured using Nikon microscope [48].
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Statistics and Data Analysis
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All experiments were performed in triplicates. Statistical analysis was performed using Instat,
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Graph Pad software, CA.
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3. RESULTS & DISCUSSION
Curcumin formed Polymeric Complexes with Eudragit® S100
When co-precipitated with Eudragit® S100 (Scheme 1), curcumin formed into a free flowing
fluorescent powder (Scheme 2), which has been characterized as non-covalent complexes
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between the curcumin and the polymer (Ora-Curcumin-S) by multiple techniques (Figure 1 and
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2). The existence of complexation or interaction between curcumin and the polymer in aqueous
solution was confirmed by passing the aqueous solution of Ora-Curcumin-S through a known
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molecular weight cut-off (10 kDa) (Figure 1) membrane. As expected, curcumin with a
molecular weight of 0.368 kDa completely passed through the 10 kDa cut-off membrane as a
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solution (10 % ethanol). Ethanol was added to make curcumin soluble in detectable amounts. In
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contrast, curcumin present in Ora-Curcumin-S is highly soluble in PBS. The soluble curcumin in
the Ora-Curcumin-S did not pass through the 10 kDa membrane in significant amounts. This is
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depicted as the pass-through ratio (ratio of the concentration of the solution after and before
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passing through the membrane), which was 0.042 for Ora-Curcumin-S and 0.981 for curcumin
solution, respectively (Figure 1). This observation shows that the curcumin in Ora-Curcumin-S
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solution may be in association with the high molecular weight Eudragit® S100 (~ 125 kDa), and
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therefore, could not pass through the 10 kDa cutoff membrane. However, when Ora-Curcumin-S
was dissolved in 100 % ethanol, instead of aqueous buffer, the pass-through ratio increased to
1.031 indicating the disruption of interactions between curcumin and the polymer. The released
curcumin dissolved in ethanol and completely passed through the 10 kDa membrane. Such
disruption of the interaction between curcumin and Eudragit® S100 was also observed with
other organic solvents such as DMSO, which indicates that the association between curcumin
and the polymer is non-covalent [41], which was further confirmed by 1H-NMR studies.
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Scheme 1. Preparation scheme of Curcumin-Eudragit® S100 complexes (Ora-Curcumin-S).
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Solid-state FT-IR spectra of curcumin (Figure 2A) displayed a broad phenolic -OH band at
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~3500 cm-1. This peak was reduced and broadened in Ora-Curcumin-S indicating the possibility
of hydrogen bonds between the phenolic -OH group of curcumin and the backbone of Eudragit®
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S100. The previous report from our laboratory depicts similar interaction pattern between
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curcumin and Eudragit® EPO, which shares the backbone structure with the Eudragit® S100
[41]. Ora-Curcumin-S also showed the peaks representing the -C=O group of Eudragit® S100
backbone. The 1H NMR spectra of Ora-Curcumin-S in alkaline D2O (0.25N Na2CO3) showed a
broad and weak signal in the aromatic region (6.0–9.5 ppm), possibly representing curcumin
(Figure 2B). However, the curcumin signal became sharper with the addition of DMSO-d6 to the
solution. Similar to the previous observation with 100 % alcohol (Figure 1), the addition of
another organic solvent (DMSO) caused the disruption of the complexes and released curcumin
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into the organic solvent that displayed sharp peaks in the NMR spectrum. The contribution of
possible hydrophobic interactions between the curcumin (aryl skeleton) and the polymer
Eudragit® S100 (alkyl chain driving the complex formation was validated by the disruption of
the complexes upon the addition of an organic solvent. (Scheme 2). Our previous studies
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demonstrated similar hydrophobic interactions between curcumin and Eudragit® EPO [41].
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Taken together, both hydrophilic and hydrophobic interactions might have contributed to the
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complex formation between curcumin and Eudragit® S100. The interactions existed both in
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solid and solution states.
Scheme 2. Structure of Curcumin and Eudragit ® S100, and light and SEM pictures of Ora-
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Curcumin-S powder.
DSC analysis of curcumin displayed a sharp endothermic peak at 187.39˚C, indicating
the melting of crystalline curcumin. Eudragit® S100 polymer (Figure 2C) did not exhibit a
distinct melting point indicating the amorphous nature of the polymer. However, we observed an
enthalpy of relaxation as an endotherm at ~126 ˚C for Eudragit® S100, which might be due to
aging or relaxation over time as observed in amorphous materials [49]. A melting peak of
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curcumin at 185.39˚C and the relaxation peak of the polymer (Eudragit® S100) at ~126 ˚C is
observed in the physical mixture of curcumin and Eudragit® S100 (Figure 2C). In contrast, OraCurcumin-S had displayed only the polymer relaxation peak; the melting peak of curcumin was
absent, which suggests that curcumin in Ora-Curcumin-S exists in an amorphous form. This was
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further confirmed by the powder-XRD spectra and the SEM image of Ora-Curcumin-S (Scheme
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2). The XRD spectra of curcumin depicted the crystalline nature of curcumin as seen from the
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appearance of characteristic peaks of curcumin (Figure 2D), whereas Eudragit® S100 was in
predominantly amorphous form with no distinct peaks (Figure 2D). In contrast to synthetic
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curcumin, distinct XRD peaks of curcumin were absent in Ora-Curcumin-S. This further
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confirms the results of DSC study that the curcumin is in amorphous form in Ora-Curcumin-S
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(Figure 2D) [41].
The amorphous form of the drug is more water soluble than the crystalline form, which is
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beneficial for poorly soluble drugs such as curcumin. In one of the popular solubility
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enhancement techniques solid dispersions, the drug exists in amorphous form along with
hydrophilic polymers such as polyethylene glycol, poly-vinyl pyrrolidone, and poly
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methacrylates. In addition, the drug may interact with the polymer in solid form [50-53]. Indeed,
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several Eudragit® polymers have been reported to form solid dispersions with poorly soluble
drugs including curcumin to enhance the drug solubility [52, 53]. Previously, our laboratory
reported that curcumin forms highly water soluble non-covalent complexes with Eudragit® EPO
(Ora-Curcumin-E) that enhanced the bioavailability of curcumin [41]. In both Ora-Curcumin-S
and Ora-Curcumin-E, curcumin formed non-covalent complexes with the polymer.
Complexation is different phenomena than solid dispersion where the interactions between the
drug and the polymer continue to exist even once dissolved.
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Figure 1. Assessment of complex formation between curcumin and Eudragit® S100
polymer (Ora-Curcumin-S). Curcumin and curcumin equivalent of Ora-Curcumin-S were
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dissolved in various solvents (A. Ora-Curcumin-S in PBS, pH 7.0, B. Ora-Curcumin-S in
ethanol, C. Curcumin in ethanol, D. Curcumin in 10% ethanol in PBS, pH 7.0). The pass-through
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ratio (bottom/top) is defined as the concentration of soluble curcumin that had passed through the
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10 kDa cut-off membrane (bottom) to the concentration before filtration (top). Data represent
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mean ± standard deviation (n=3-4). Complexation with the Eudragit® S100 with ~125 kDa size
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prevented curcumin to filter through the 10kDa cutoff membrane.
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Figure 2. Physicochemical characterization of Ora-Curcumin-S. 2A) FTIR spectrum of
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curcumin (in blue), Eudragit® S100 polymer (in red), curcumin and Eudragit® S100 physically
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mixed (in cyan) and Ora-Curcumin-S (in green). A band representing the phenolic –OH group in
curcumin is diminished in Ora-Curcumin-S (in green). 2B) 1H NMR spectrum of Ora-
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Curcumin-S (30 mg) dissolved in basic D2O before (curve A) and after the addition of equal
volume of DMSO-d6 (curve B) 2C) DSC analysis curves of curcumin (a), Eudragit® S100 (b),
curcumin and Eudragit® S100 physical mixture (c), and Ora-Curcumin-S (d). 2D) X-ray
diffraction patterns (XRD) of powders of curcumin (a), Eudragit® S100 (b), a physical mixture
of curcumin and Eudragit® S100 (c), and Ora-Curcumin-S (d). The x-axis represents 2ɵ degree
and Y-axis represents intensity in (cps).
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Formulation Optimization of Ora-Curcumin-S
To enhance the drug loading onto the polymer, preparation of Ora-Curcumin-S was optimized by
altering various formulation parameters: the type of solvent, type of surfactant/stabilizer and the
drug to the polymer ratio. Higher loading of the drug is necessary to reduce the total dose of Ora-
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Curcumin-S during preclinical or clinical testing. The drug loading and aqueous solubility are
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dependent upon the non-covalent interactions between the curcumin molecules (intramolecular
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interactions) and the interactions between the curcumin and the polymer molecules
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(intermolecular interactions) during complexation. These interactions are influenced by the
polarity of the organic solvent used during co-precipitation, and hence, the solvents were
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selected based on their polarity/dielectric constant (Acetone-21; Ethanol-24.5; Methanol-33 and
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DMSO-46.7). An enhanced curcumin loading (152.84 ± 1.55 µg/mg for 1:2 drug to polymer
ratio) was achieved when acetone + DMSO (7:3) was utilized as a solvent as compared to other
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organic solvents evaluated (Table 1). The type of stabilizer was also altered in a subsequent set
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of studies (Table 1). When PVA was utilized as a stabilizer, the loading of curcumin was
increased significantly as opposed to Pluronic F-68 or Tween 20 as stabilizers. Changing the
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drug to polymer ratio (Table 2) also influenced the drug loading. The highest loading of ~150
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µg/mg of curcumin was achieved with acetone + DMSO as the solvent, PVA as the stabilizer and
1:2 curcumin to Eudragit® S100 ratio as formulation parameters. The changes in the formulation
did not alter the complexation behavior or reduced the apparent solubility of Ora-Curcumin-S in
PBS.
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Table 1. Formulation optimization of Ora-Curcumin-S. Effect of various formulation factors
during the preparation of Ora-Curcumin-S on the amount of curcumin loading and apparent
solubility in PBS, pH7.4 (in 4 h). Data represent mean ± standard deviation (n=3-4). ***
indicates that the values are significantly higher compared to all other groups (p ≤ 0.001)
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calculated using one-way ANOVA followed by Dunnett’s post-hoc multiple comparison tests.
times) by Ora-Curcumin-S technology.
Surfactant/Stabilizer
PluronicF68
Tween-20
PVA
PluronicF68
Tween-20
PVA
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Ethanol
1:2
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Acetone
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PVA
PluronicF68
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Methanol
Curcumin:
Eudragit®
S100 ratio
1:2
1:2
Tween-20
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PVA
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Acetone+DMSO
PluronicF68
Tween-20
Curcumin
Loading
(µg/mg)
Apparent
Solubility
(mg/ml)
115.50 ± 6.01
0.539 ± 0.057
89.41 ± 9.09
96.57 ± 7.58
45.60 ± 1.18
66.38 ± 4.74
131.0 ± 14.60
50.02 ± 1.81
0.582 ± 0.014
0.267 ± 0.013
0.068 ± 0.051
0.056 ± 0.017
0.373 ± 0.030
0.234 ± 0.041
43.84 ± 0.52
0.234 ± 0.029
59.21 ± 2.31
0.060 ± 0.016
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Organic
Solvent
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The apparent solubility of curcumin (<1 µg/ml) [30] was enhance up to ~1000 µg/ml (~1000
152.84 ± 1.55
1:2
***
140.6 ± 6.51
85.44 ± 7.79
1.093 ± 0.27
***
0.653 ± 0.046
0.291 ± 0.099
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Table 2. The effect of curcumin to Eudragit® S100 ratio on the loading of curcumin. OraCurcumin-S prepared using acetone-DMSO as organic solvent and PVA as a stabilizer. Data
represent mean ± standard deviation (n = 3).
Curcumin
loading (μg/mg)
1:20
1:10
1:5
1:2
3.01 ± 0.29
23.11 ± 5.46
50.54 ± 6.91
152.84 ± 1.55
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Curcumin:
Eudragit® S100 ratio
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Ora-Curcumin-S is Water Soluble and Stable form of Curcumin
Ora-Curcumin-S is specifically designed for the local treatment of IBD or colon cancer.
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Therefore, it is very important for an active component to remain stable during its transit through
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the GI tract and to be soluble for producing local effects only when it reaches to the colon. The
apparent (kinetic) solubility of Ora-Curcumin-S was estimated in phosphate buffer, pH 7.0 for 4
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h. The soluble curcumin was separated and estimated. The aqueous solubility of unformulated
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curcumin (<0.001 mg/ml) was significantly enhanced (~1000 times) when delivered as OraCurcumin-S (Table 1). Physical addition of curcumin to blank Eudragit® S100 complexes could
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not significantly enhance the solubility of curcumin. Blank Eudragit® S100 complexes were
prepared by precipitating Eudragit® S100 without curcumin in the presence of PVA. This
observation suggests that the enhanced solubility observed with Ora-Curcumin-S is not due the
presence of formulation components; however, the complex formation is necessary between the
drug and the polymer. Similar to the loading, the highest apparent aqueous solubility was also
observed with the following formulation parameters; acetone+DMSO as a solvent, PVA as a
stabilizer, and with 1:2 drug polymer ratio (1.09 ± 0.27 mg/ml). Since, the drug needs to be in
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soluble form for eliciting biological activity, enhancing the aqueous solubility of poorly soluble
curcumin will have positive therapeutic implications in the colon.
Curcumin is highly unstable in aqueous buffers especially at neutral and alkaline pH,
which creates another major bottleneck for the oral delivery of curcumin [41]. The aqueous
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stability of Ora-Curcumin-S was evaluated for 24 h in phosphate buffer, pH 7.0 or simulated
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intestinal fluid (pH 6.7-6.9) along with 10 % methanol. Ten percent methanol enhanced the
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solubility of synthetic curcumin in aqueous buffers to obtain sufficient initial concentration for
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enabling the detection of curcumin even after ~80-90% degradation. It was ensured that the
complex formation between curcumin and Eudragit® S100 did not significantly alter due to the
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addition of 10% methanol as tested by the method described in Figure 1. The unformulated
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curcumin degraded by 70% within the first 2 h of the incubation. To the contrary, Ora-
and PB, pH 7.0 (Figure 3C).
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Curcumin-S was more stable with ~85-90% remained intact after 24 h of incubation, both in SIF
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Ora-Curcumin-S is a “Targeted Local Drug Delivery System” for Ulcerative Colitis
Ora-Curcumin-S is specifically designed to target the inflamed tissue at the luminal side of the
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colon through two different, however, unique strategies based on the physiological and
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pathological differences of the target tissue. A) Ora-Curcumin-S is soluble only at pHs above
6.8 (Figure 3A & B) similar to its precursor polymer Eudragit® S100. The results from an invitro drug release study in a buffer with gradually changing pH depict that a large proportion of
soluble curcumin (~80 %) was released into the medium from the Ora-Curcumin-S only when
the pH of the medium reached above 6.7 (Figure 3B). Therefore, once consumed orally, OraCurcumin-S is not expected to dissolve until the GI pH reaches around 6.8, i.e. only close to the
colon, thereby, significantly reducing the systemic absorption of the drug from the intestine
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(Figure 3D). This hypothesis was tested in a preliminary pharmacokinetic study in mice, which
showed that 1 h after the administration of Ora-Curcumin-S, there was no detectable amounts of
curcumin in the blood (Figure 3E), however, 90 % of the curcumin present in the freshly
collected fecal matter after 16 h of administration is highly water soluble (extracted with the
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phosphate buffer, pH7.0) (Figure 3F). One-hour time point for plasma levels was selected based
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on our previous observation that the time to reach maximum plasma concentration (Tmax) for
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curcumin is ~1 h. [54]. In contrast, in mice administered with unformulated curcumin, there were
undetectable levels of plasma curcumin, and importantly, 100% of the fecal curcumin was water-
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insoluble, which was extracted only with an organic solvent acetone (Figure 3E&F). Presence of
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soluble curcumin in the fecal matter is an indicative of the availability of the bioactive drug for
local activity at the colon. Ora-Curcumin-E (soluble curcumin complexes with Eudragit® EPO)
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is another technology that has been shown to enhance the absorption of curcumin, but it is not
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targeted to the colon [32, 54]. It is used as a positive control in this study for plasma levels. B)
Ora-Curcumin-S is expected to be retained longer at the inflamed sites of the colon. The contents
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of the colon stay longer to pass, unlike intestine; therefore, the inflamed tissue will be exposed to
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soluble anti-inflammatory Ora-Curcumin-S for prolonged periods of time. Evidence from studies
also reports that there is colonic mucosa inflammation complemented by the depletion of the
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mucus layer and the accumulation of positive charge at the epithelial surface. The accumulation
of positive charge could be due to the in-situ collection of proteins such as transferrin [55, 56],
permeability-increasing protein, and anti-microbial peptides which are positively charged [5759]. The exposed positive charge provides a unique molecular target for anchoring drug carriers
with negative surface charge [55, 60]. Ora-Curcumin-S is designed to carry a negative charge at
the luminal pH with the selection of Eudragit® S100 as a polymer, which has carboxylic acid
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side-chain groups (Scheme 2). Therefore, it is expected to anchor the damaged epithelial cells at
the inflammation site through charge-based interactions to provide prolonged contact time for
better uptake of the drug and improved therapeutic outcomes. Such strategies have been
previously reported to improve the ability of drugs to target inflamed tissue in IBD models [55,
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60, 61]. Taken together, Ora-Curcumin-S is a unique delivery technology that significantly
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reduces the exposure of bioactive curcumin to systemic circulation or metabolic enzymes,
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however, boosts the soluble curcumin levels in the lumen of the colon.
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Ora-Curcumin-S Enhanced the Intracellular Delivery of Curcumin
Ora-Curcumin-S represents highly soluble and stable form of curcumin and therefore, it
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enhanced the intracellular delivery of curcumin. The uptake of Ora-Curcumin-S into the human
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colon cancer cells, HT29 and HCT116 were compared with synthetic curcumin at pHs 5.5 and
7.0. The pH 5.5 and 7.0 represents the luminal pHs of the proximal and distal intestine. After 4 h
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of incubation, Ora-Curcumin-S was highly efficient in delivering the curcumin to the colon
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cancer cells (Table 3). Ora-Curcumin-S on average delivered ~10 times (HT29 cells) and ~ 3
times (HCT116 cells) more curcumin to each cell compared to synthetic curcumin when
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delivered at pH 7.0 (Table 3). Intracellular delivery of Ora-Curcumin-S is pH-dependent. The
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efficiency of delivery is very high at pH 7.0 compared to pH 5.5, which is expected as OraCurcumin-S soluble only at pHs > 6.7 (Figure 3A & B).
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Figure 3. 3A) The pH-dependent solubility of Ora-Curcumin-S: The concentration of soluble
curcumin as depicted in solutions at various pHs (1.2, 4.5, 7.0 and 7.4). 3B) The in-vitro release
profile of curcumin from Ora-Curcumin-S incubated in a buffer that gradually changed pH over
16 h (simulating GI transit time). Data represent mean ± standard deviation (n=3-4). 3C)
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Aqueous stability of Curcumin or Ora-Curcumin-S in phosphate buffer, pH 7.0 and simulated
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intestinal fluid (pH 6.7-6.9) for 24 h. Data represent mean ± standard deviation (n=3-4). 3D-F)
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Colon-targeted delivery of Ora-Curcumin-S (D). The plasma curcumin concentration (1 h) (E)
and the proportion of soluble curcumin in the colon contents (16 h) (F) after oral administration
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of various curcumin formulations at 15 mg/kg dose. The absence of detectable amounts of
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curcumin in the plasma (E) and the presence of high proportion of soluble curcumin in the
freshly collected fecal pellet, (F) are evident in Ora-Curcumin-S administered mice. Ora-
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Curcumin-E is a positive control for plasma concentration.
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Table 3. Intracellular delivery of curcumin. Intracellular delivery of curcumin by synthetic
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curcumin and Ora-Curcumin-S at various intestinal pHs in HT29 and HCT116 cells measured
after 4 h of incubation with the formulations. The amount of curcumin delivered was quantified
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using flowcytometry. The data represent mean ± standard deviation (n = 3). *** indicates that
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the values are significantly higher compared to curcumin treatment (p ≤ 0.001) calculated using
one-way ANOVA followed by Bonferroni’s post-hoc multiple comparison tests.
Mean Fluorescence Intensity (MFI)
HT29
Groups
Curcumin
Ora-Curcumin-S
HCT116
pH 5.5
pH 7.0
pH 5.5
pH 7.0
2704.67 ± 221.62
2429 ± 247.47
2543 ± 72.52
3514 ± 975.67
7188.33 ±
1398.27***
20998.67 ±
2210.80***
3555.67 ±
1168.97
8783.67 ±
1760.08***
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Ora-Curcumin-S is a Novel Polymer-Based TLR-4 Antagonist
Recently, curcumin has been identified to alter the function of TLRs [13, 62]. The TLR-4
modulating activity of Ora-Curcumin-S was assessed by its ability in inhibiting the MPLA or E.
coli (TLR-4 agonists) induced an inflammatory response in DC2.4 and HEKTLR-4YFPMD2 cells.
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Ora-Curcumin-S (5 μg/ml) was significantly more potent than solubilized curcumin (in DMSO)
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in inhibiting the secretion of TNF-α by DC2.4 cells in response to known TLR-4 agonists
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(MPLA, 2 µg/ml (Figure 4A) and E.Coli, 5x105 cells/ml) (Figure 4B). Use of E.Coli instead of
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LPS signifies the microbe induced immune-stimulation in IBD. Dendritic cells were chosen for
these studies as they express multiple TLRs and regulate the innate immunity against pathogens
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and tolerance towards commensals along with macrophages by impacting T-cell differentiation
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during the mucosal inflammation [63]. Further, the key role of TNF- in IBD pathology is wellestablished and inhibiting TNF-α has been an active strategy for the therapeutic intervention of
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IBD (Ex. Remicade, Humira, Cimzia, etc.). The TLR-4 antagonist activity of Ora-Curcumin-S
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was further validated using HEKTLR-4YFPMD2 cells over-expressing exclusively TLR-4 among
all the TLRs. These cells release IL-8 as a result of downstream signaling cascade upon the
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activation of the TLR-4 that is expressed on their surface [43]. Ora-Curcumin-S enhanced the
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activity of soluble curcumin (in DMSO) significantly in antagonizing the stimulation of TLR-4
by MPLA in these cells at equivalent curcumin concentrations of more than 5 µg/ml (Figure 4C).
Multiple factors including increased aqueous stability, improved cellular uptake or enhanced
receptor binding might have contributed to the higher TLR-4 antagonistic activity of OraCurcumin-S.
Accumulating evidence strongly suggests the critical role of TLRs-MyD88 signaling in
the etiology of IBD and colon cancer [18]. In this regard, several studies using genetic,
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physiological and pharmacological models have now established that the deregulation of TLR4
and associated signaling promote IBD severity and also susceptibility to colon cancer [15-17, 64,
65]. Physiologically, altered TLR4 signaling has been linked to the changes in mucosal barrier
properties [19, 21, 22], intestinal microbiota [22, 23], and immune activation [64]; key changes
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connected to the IBD or colon cancer. Indeed, TLR4 has been identified as a highly effective
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therapeutic target to minimize IBD-associated morbidity and susceptibility to colon cancer [13,
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23, 66]. Therefore, targeted delivery of a polymer based TLR-4 antagonist (Ora-Curcumin-S) to
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the inflammation site in IBD will have higher translational value as a new therapeutic option.
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Figure 4. Inhibition of TLR-4 Activity. Mouse dendritic cells (DC2.4) were pre-incubated with
either solubilized curcumin in DMSO or curcumin equivalent of Ora-Curcumin-S at 5 and 10
μg/ml concentrations for 1 h before activating TLR-4 by the addition of MPLA (2 μg/ml)
(Figure 4A) or E.Coli (5×104 cells) (Figure 4B). After 48 h, the levels of TNF-α in the cell
supernatants was estimated by ELISA as a pro-inflammatory marker. The data represent
mean ± standard deviation (n = 3). *** indicates that the values are significantly higher
compared to all other groups (p ≤ 0.001) calculated using one-way ANOVA, followed by
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Bonferroni’s post-hoc multiple comparison tests. Figure 4C represents the dose-response curve
of solubilized curcumin and equivalent of Ora-Curcumin-S on inhibiting MPLA (2 μg/ml)
induced TLR-4 activation on HEK293TLR4YFP-MD2 cells as represented by IL8 levels in the
supernatant. Figure 4D represents an increased resistance of HEK293TLR4YFP-MD2 cells
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against MPLA-induced activation of TLR-4 signaling in the presence of curcumin or curcumin
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equivalent of Ora-Curcumin-S at fixed 5 μg/ml concentration. In both Figure 4C and 4D, the data
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represents mean ± standard deviation (n = 3). *** indicates that the values are significantly
different (p ≤ 0.001) compared to solubilized curcumin group calculated using one-way
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ANOVA, followed by Bonferroni’s post-hoc multiple comparison tests.
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Ora-Curcumin-S Reduced the Proliferation of Colon Cancer cells
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Inadequately managed IBD significantly increases the risk of developing colon cancer. In
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addition, curcumin possesses anti-cancer potential. Therefore, the effect of curcumin
formulations on human colon cancer cells (HT29 and HCT116) was investigated using the MTT
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assay. Ora-Curcumin-S was dissolved in PBS, pH 7.0. Since curcumin is water insoluble, two
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different stocks were prepared either by dissolving curcumin in DMSO and dispersing in PBS,
pH 7.0 buffer. As expected, dispersed curcumin in PBS did not suppress the proliferation of
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HT29 and HCT116 cells, as there was very little soluble curcumin to enter the cells (Figure 5A
& B). However, both the soluble curcumin formulations suppressed the growth of colon cancer
cells in a dose-dependent manner (Figure 5A & B). Compared to solubilized curcumin (IC50 for
HT29 41.84 ± 1.86 M and HCT116 18.35 ± 1.94 M), Ora-Curcumin-S (IC50 for HT29 17.47 ±
0.63 M and HCT116 10.39 ± 1.08 M) showed a significant reduction in cell viability. The
stronger inhibitory effect of Ora-Curcumin-S could be attributed to an enhanced intracellular
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delivery through Ora-Curcumin-S, which may be because of improved aqueous solubility and
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stability Ora-Curcumin-S.
Figure 5. Effect of curcumin or curcumin equivalent Ora-Curcumin-S on cell viability of
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colon cancer cells. Two different colon cancer cells of human origin, HT29 (Human colorectal
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adenocarcinoma) (Figure 5A) and HCT116 (Human colorectal carcinoma) (Figure 5B) were
treated with varying concentrations (0-40 µM) of either curcumin (in pH7.0 buffer or DMSO) or
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curcumin equivalent of Ora-Curcumin-S (in pH 7.0 buffer). After 48 h, the cell viability was
estimated by the MTT assay. The percent viability normalized to untreated cells is represented
against the concentration of the drug. The data represent mean ± standard deviation (n=4-5). *
and § indicates that the values are significantly higher compared to curcumin group in pH 7.0
buffer and curcumin in DMSO respectively (p ≤ 0.001) calculated using two-way ANOVA
followed by Bonferroni’s post-hoc multiple comparison tests. Data represent mean ± standard
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deviation (n=4-5). Note: Synthetic curcumin was not soluble in pH 7.0 buffer, and therefore, did
not significantly alter the viability of the cells.
The Effect of Ora-Curcumin-S on DSS-induced Ulcerative Colitis in mice: A proof-of-concept
To test the clinical efficacy of the in-vitro findings that Ora-Curcumin-S could be a potent
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polymer-drug/delivery system for localized treatment to inhibit mucosal inflammation and
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associated injury, we employed a widely utilized murine model of ulcerative colitis, where mice
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are administered dextran sodium sulfate (DSS) in drinking water for a period of 7 days. In
addition to the ease of performing these studies and reproducibility, this model produces
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pathology similar to the human ulcerative colitis as genes upregulated in DSS-colitis and human
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ulcerative colitis are highly identical [47, 48]. Mice (C57BL/6J strain, 8-10 weeks old) were
exposed to DSS (2.5% w/v in drinking water) for seven consecutive days with or without oral
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administration (gavage) of Ora-Curcumin-S.
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Ora-Curcumin-S Ameliorates Intestinal Inflammation and Associated Injury in Mice
Subjected to DSS-Colitis: The results from the study demonstrated that the mice treated with
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DSS alone showed significant body weight loss day 5 post DSS administration compared to the
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mice fed on regular drinking water without the DSS (p < 0.01). However, in mice receiving OraCurcumin-S in addition to the DSS, body weight remained relatively stable and the weight loss at
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the time of sacrifice was significantly lower compared to the DSS-treated mice (p < 0.01)
(Figure-6A). The cumulative clinical disease index, based on scoring of diarrhea, rectal bleeding
etc., further showed less severe clinical disease in DSS+Ora-Curcumin-S group compared to the
DSS treated mice (p < 0.01) (Figure-6B). The colonoscopic evaluation at day 5 post DSSadministration showed a signifiacant reduction in inflammation and injury in mice receiving
DSS+Ora-Curcumin-S versus DSS alone (Figure-6C). Results demonstrated that the colon length
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in DSS+Ora-Curcumin-S mice was significantly increased (p < 0.001; Figure-6D-E) while
edema (thickness) and spleen weight were significantly lower (p < 0.05; Figure-6F-G). In the
blinded H&E evaluation by an experienced gastrointestinal/pathologist, DSS treated colon
exhibited clear signs of inflammation including immune cells infiltration, loss of goblet cells,
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crypt damage and erosion or irregular mucosal structure (Figure-6H). In comparison, DSS+Ora-
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Curcumin-S group demonstrated significantly less score for the cumulative injury compared to
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DSS treated mice (Figure-6I and Supplementary Figure-3). Moreover, Periodic acid schiffhaematoxylin (PAS) staining demonstrated that the DSS+Ora-Curcumin-S treated mice colons
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possessed significant proportion of the regenerative colonic crypts with mucus producing goblet
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cells morphology (Figure 7F), which was closer to the healthy colon than the colon from mice
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subjected to colitis.
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Figure 6. Ora-Curcumin-S promotes mucosal healing during DSS induced colitis. The
colitis was induced in mice by administration of DSS (2.5% w/v) in the drinking water for 7
consecutive days. (A) & (B). The body weight (represented in terms of %) and Disease activity
index (DAI) changes during the course of 7 days for the DSS alone and DSS+Ora-Curcumin-S
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treatment respectively. (C). The colonoscopic analysis shows no sign of inflammation in
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DSS+Ora-Curcumin-S treatment group compared to DSS alone. (D-E). Representative colon
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images and colon length (cm) in control, DSS-treated versus DSS + Ora-Curcumin-S group; (F).
Colon edema evaluated as weight/cm decreases with Ora-Curcumin-S treatment; (G). DSS+Ora-
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Curcumin-S treatment reduces the spleen weight versus DSS treated mice; (H). Haematoxylin
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and eosin stained representative images of the colonic tissues from control, DSS-treated, and
DSS+Ora-Curcumin-S treatment group mice; (I). Cumulative injury scores. Ora-Curcumin-S
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treatment group mice show reduced inflammation and associated injury. Values are presented as
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a mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001.
Ora-Curcumin-S Reduces the Pro-Inflammatory Signaling Induced by DSS colitis:
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Considering the key role of the pro-inflammatory signaling including TNF-α activation in IBD
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and results from in-vitro studies that Ora-Curcumin-S inhibits the TLR-4 signaling, we further
examined if protective effects of the Ora-Curcumin-S administration are mediated by inhibiting
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the pro-inflammatory cytokines expression and/or inducing adaptive immunity. Analysis using
qRT-PCR demonstrated that mRNA expression of the pro-inflammatory cytokines TNF-α and
IL-6 sharply increased in response to DSS exposure. However, these increases were blunted (p <
0.05) in mice that received Ora-Curcumin-S along with the DSS (Figure-7A-B). In contrast, IL10, well-known for its anti-inflammatory properties, was significantly increased (p < 0.001) in
DSS colitis as reported by other studies, however, increased further (p < 0.05) in DSS+Ora-
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Curcumin-S treated mice (Figure-7C). Encouraged by the above findings and the fact that TNFα and IL-6 help induce NF-kB signaling, the rheostat of the mucosal inflammation, we examined
if Ora-Curcumin-S treatment also reduced the NF-kB signaling, the pivotal signaling in
promoting mucosal inflammation. Immunohistochemistry analysis was done using the anti-P65
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antibody as nuclear immunoreactivity of this antibody supports NF-kB activation. Indeed, we
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found significantly reduced nuclear localization of the NF-kB in Ora-Curcumin-S treated mice
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compared to DSS treated group (Figure 7D). Taken together, Ora-Curcumin-S demonstrated
significant and promising effects in inhibiting the pro-inflammatory cellular processes and
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promoting mucosal healing (Figure 7A-F).
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Figure 7. Ora-Curcumin-S reduces the pro-inflammatory signaling and restores the colonic
mucosa. Total RNA was subjected to qRT-PCR to examine the expression of pro-inflammatory
cytokines. Ora-Curcumin-S treatment along with DSS demonstrates suppression of these proinflammatory cytokines: (A). TNF-α, (B). IL-6 while increases the anti-inflammatory cytokine
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(C). IL-10; (D). Immunohistochemical analysis showing reduced nuclear localization of p65
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protein during Ora-Curcumin-S treatment; (E). Ora-Curcumin-S treatment increases the Mucin-2
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production to protect colonic mucosa during DSS colitis; (F). PAS staining showing that OraCurcumin-S treatment increases mucin producing goblet cells and helps in restoring of colonic
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mucosa. Values are presented as a mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001.
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Ora-Curcumin-S is a Unique Delivery Technology Compared to Previously Reported
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Curcumin Formulations
The interest in designing novel curcumin formulations increased significantly after the failure of
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several curcumin clinical trials due to its poor pharmacokinetic profile [27]; both in the
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pharmaceutical industry and in the academia reviewed at [28, 67-69]. The majority of the
reported or marketed curcumin formulations come under one of the following broad strategies,
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which are discussed below. A) Unformulated Curcumin: which is practically water insoluble
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(~0.001 mg/ml). Therefore, it will neither absorb into the blood circulation nor provide soluble
active curcumin at the lumen of the colon (Figure 8A). B) Bioavailable Curcumin: Majority of
the curcumin formulations reported are targeted to enhance the oral absorption of the curcumin
to increase its bioavailability (Figure 8B). However, they still fall short in maintaining the
sustained therapeutic plasma levels as the improved absorption was negated by the rapid
elimination of curcumin (half-life only in minutes) [25, 70] and causes metabolic interactions
with the other drugs [40]. C) Colon-Targeted Curcumin: Numerous traditional therapeutics for
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IBD are delivered specifically to the colon using delayed release coating-based technologies
(Asacol®, Entocort EC®, Lialda® etc.). Using similar technologies, few recent studies have
reported the delivery of curcumin to the colon for treating IBD or colon cancer [44, 71-75].
These studies are based on nanoparticles loaded with curcumin and coated with polymers
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including Eudragits® to delay the release of curcumin from nanoparticles. They are competent in
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delaying the onset of drug release to bypass the portions of the intestine. However, without
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significantly enhancing its solubility, curcumin will not provide a high soluble concentration in
the colon (Figure 8C). In contrast; Ora-Curcumin-S is unique with the following three main
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advantages. a) In addition to avoiding the absorption into the systemic circulation, similar to
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delayed-release formulations, highly soluble (>2 mg/ml) Ora-Curcumin-S will provide soluble
and bio-active fraction in the colon contents (Figure 3D) b) Being negatively charged, Ora-
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Curcumin-S is expected to retain better at the positively charged inflammation site. c) In
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addition, Ora-Curcumin-S is polymer based complexes which inhibits the TLR-4 signaling
(Figure 4A-D), which is linked to the inflammation in colitis and colitis-induced cancer. In
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addition, the procedure to prepare Ora-Curcumin-S is simple (precipitation in acid water) to scale
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up compared to nanotechnologies and controlled coating techniques. The materials (curcumin,
Eudragits, and polyvinyl alcohol (PVA)) are economical and have received GRAS certification
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(Generally Regarded as Safe) from the US-FDA. The reasons for utilization of GRAS reagents
safe for oral consumption are because they are inexpensive, readily available in sufficiently
larger amounts. This would accelerate the translation into the clinic.
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curcumin formulations.
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Figure 8. Ora-Curcumin-S is a unique technology compared to previously reported
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4. CONCLUSION
A polymer based water soluble TLR-4 antagonist (Ora-Curcumin-S) was designed by
complexing curcumin with Eudragit® S100 that has a unique ability to target the luminal side of
the colon for inflammatory bowel disease such as ulcerative colitis. This proof-of-concept study
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will provide a strong basis for further preclinical and clinical analysis for the use of Ora-
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Curcumin-S in inhibiting IBD severity and colitis associated cancer. Ora-Curcumin-S has several
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competitive advantages over existing technologies: high aqueous solubility & loading, local
delivery, high aqueous stability, prevention of IBD, cost effective and use of FDA approved
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ingredients etc. This study advances the therapeutic application of Ora-Curcumin-S in various
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complex chronic diseases such as IBD & colon cancer. As an anti-bacterial and by altering host
immune system to control inflammation, Ora-Curcumin-S is also expected to modulate colon
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microbiome and improve GI health. In addition, the use of safe, established materials along with
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simple procedure will expedite its translational path from bench research to bedside clinical
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application.
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SUPPORTING INFORMATION
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Supplementary data relevant to the study has been included in the supporting information.
ACKNOWLEDGEMENTS:
The authors would like to acknowledge the Translational Cancer Research Center, Office of
Research (Faculty Excellence Grant), SDSU and the Department of Pharmaceutical Sciences,
SDSU for providing the funds for the study. We acknowledge Mr. Pratik Muley for his
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assistance during the initial formulation development. The support from the Sanford ProfileBOR Innovation Grants, South Dakota (2016) and the Grant-In-Aid of Research from Sigma Xi
for Mr. Kesharwani, a graduate student is gratefully acknowledged. We thank Dr. Mohit Tyagi
for his assistance during NMR experiment and interpretation of the NMR data. The results of
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flow cytometry were obtained by utilizing the Sanford Research Flow Cytometry Core Facility.
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This is supported in part by a COBRE grant from the National Institutes of Health (P20
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GM103548). We would like to acknowledge Mrs. Maudi Killian Marisela for her assistance with
the flow-cytometry experiments and the data analysis related to it. This work was supported by
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DK088902 (NIH) and BX002761 (VA-merit award) (Amar. B. Singh).
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Notes/Competing financial interests
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Siddharth S. Kesharwani and the corresponding author Dr. Hemachand Tummala are inventors
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on the patent application filed related to this technology.
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