Abstract
Cyclodextrin complex of nintedanib was prepared aiming for increased bio-activity and improved transport across intestinal membrane with reduced p-glycoprotein (p-gp) efflux. Based on preliminary phase solubility studies and molecular modeling, sulfobutyl ether derivative of βcyclodextrin (SBE-β-CD, Captisol®) was selected to prepare inclusion complex. Complexation was confirmed using FTIR, 1H NMR, DSC, and XRD.Bioactivity of the formed complex was tested using lung fibroblast cells, WI-38 for anti-proliferative activity and effect on collagen deposition and cells migration. In-vitro permeability studies were performed using epiIntestinal tissue model to assess the effect of complexation on transport and p-gp efflux. Results of the study demonstrated that cyclodextrin complexation increased stability of nintedanib in PBS (pH 7.4) and simulated intestinal fluid (SIF).Further, bioactivity of nintedanib also improved.
Interestingly,complexation has increased transport of nintedanib across intestinal membrane and reduced efflux ratio, suggesting the role of cyclodextrin complexation in modulating pgp efflux.
Keywords: Nintedanib, cyclodextrin, intestinal permeability, pglycoprotein efflux, pulmonary fibrosis
1. Introduction:
Nintedanib, akinaseinhibitor, has been approved by the Food and Drug Administration (FDA) for treatment of idiopathic pulmonary fibrosis (IPF) (Mazzei, Richeldi, & Collard, 2015). It inhibits multiple receptor tyrosine kinases and non-receptor tyrosine kinases (Richeldiet al., 2014; Vaidya, Patel, Muth, & Gupta, 2017), including platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR) and Fms-like tyrosine kinase-3 (FLT3) (Rothetal., 2015). PDGFR, FGFR and VEGFR have been reported to play a critical role in the pathogenesis of IPF (Chaudhary et al., 2007; Coward, Saini,& Jenkins, 2010; Richeldi et al., 2014). Nintedanib (Vargatef®) has also been
approved, in combination with docetaxel, by the European Agency Medicines (EMA) for the treatment of nonsmall cell lung cancer (NSCLC) (Espinosa Bosch, Asensi Diez, Garcia Agudo, & Clopes Estela, 2016). It is also in clinical trials to be used as single agent or in combination with other chemotherapeutic agents as first-line treatment for a variety of cancers, including breast, ovarian, NSCLC, brain are among some of them (www.clinicaltrials.gov). Currently, nintedanib is available by prescription as oral capsules (Ofev® and Vargatef®) and is recommended to use for twice daily dosing. Oral bioavailability of the 100 mg nintedanib oral capsules was reported to be approximately 5% and is believed to be significantly low due to substantial first-pass metabolism and efflux by transporter pumps (Dallingeret al., 2016). Nintedanibis known to be a substrate for p-glycoprotein (p-gp) and it was reported clinically that p-gp inhibitors increased the bioavailability of nintedanib when administered simultaneously in healthy subjects (Luedtke,Marzin, Jungnik, von Wangenheim, & Dallinger, 2018).
Cyclodextrins (CDs) are cyclic polysaccharides consisting of 6-8 D-glucose monomers linked by α1,4-glucosidic bonds. The structures of CDs are arranged in such away that they have hydrophobic cavities with hydrophilic outer surfaces. Due to the presence of hydrophobic cavity, CDs have capability to form inclusion complex with less water soluble compounds, which are retained in the cavity of CDs by different molecular interaction (Davis & Brewster, 2004; Yang, Lin, Chen, & Liu, 2009). Among different CDs, β-CDs are most commonly used because of their low price, easy availability and structural orientation favorable for inclusion complex formation; and are well reported to improve in-vivo stability and bioavailability of small molecules (Lima et al., 2016). Increase in the bioavailability of CD-drug inclusion complex has been reported to occur by different mechanisms including solubility enhancement, increasing stability of the compounds in the intestinal environment, increasing the interaction with cell membrane, decreasing the barrier function of lipophilic membrane, by modulating p-gp activity, and combinations of these mechanisms (Loftsson, Jarho, Masson, & Jarvinen, 2005; Masson, Loftsson, Masson, & Stefansson, 1999; Nakanishi, Nadai, Masada, & Miyajima, 1992; Rong et al., 2014; Zhang, Cui, Gao, & Jiang, 2013). While the mechanisms for p-gp activity modulation by CDs are not clear, results of different studies reveal following point (Zhang et al., 2013). CDs are poor substrates for p-gp because these are hydrophilic, neutral, and high molecular weight compounds with less cell permeability. It has also been suggested that CDs (specially lipophilic dimethyl-β-CD) release pgp transporters from the apical membrane by depleting cholesterol from the cell membrane and thus reduce the p-gp function of intestinal epithelial cells (Yunomae, Arima, Hirayama, & Uekama, 2003). However, it is to be noted that hydroxypropyl-β-CD and sulfobutyl ether-β-CD show less cholesterol depletion activity compared to methyland dimethyl-β-CD and inhibition of P-gp ATPase activity is demonstrated to be a mechanism of p-gp function rather than changing the cell membrane fluidity (Zhang, Meng, Cui, & Song, 2011). Hence, two both HPβ -CD and SBEβ -CD are considered to be safe in terms of cytotoxicity/hemotoxicity and nephrotoxicity (Nagase et al., 2003; Rajewski et al., 1995; Wang et al., 2011).
It has also been reported earlier that many compounds have shown improved biological activity after encapsulation in β-CDs (Dandawate et al., 2012; Nguyen, Liu, Zhao, Thomas, & Hook, 2013; Pinho, Grootveld, Soares, & Henriques, 2014; Yee et al., 2017). While nintedanib has been approved by the US-FDA for last 4 years,there are no studies reported demonstrating a methodology, either cyclodextrin complexation or any other novel carriers, to overcome known pgp efflux and in-turn reduced oral bioavailability. In the present study, we hypothesize that by forming inclusion complex with CD, permeability/bioavailability of nintedanib could be improved by increasing stability in intestinal tract and also by modulating p-gp efflux. Further, bio-activity of nintedanib could also be improved by forming inclusion complexes with cyclodextrins.
2. Materials and Methods
Nintedanib free base (>99%) and nintedanib ethanesulfonate (EHS) salt (>99%) were purchased from LC Laboratories, Woburn, MA. Sulfobutylether-β-CD (SBE-β-CD, Captisol®, average mol. wt. 2163, average degree of substitution=6.6) was provided as a gift sample from Cydex Pharmaceutical Inc. KS, USA. Hydroxypropyl-β-CD (HPβ-CD, Cavasol®, W7 HP, average mol. wt. 1410, degree of substitution =4.1-5.1) was purchased from Ashland (produced by Wacker Chemie AG, Burghausen, Germany). HPLC/LCMS grade solvents, and other reagents and chemicals were obtained from Fisher Scientific.
2.2 Phase Solubility Studies
Phase solubility studies were done according to previous reported method (Higuchi & Connors, 1965). Briefly, an excess amount of nintedanib base was added to aqueous solution of CDs having different concentrations (0-200 mM). Suspensions were bath sonicated for 30 min and left for 24 hours under continuous stirring for equilibration. After 24 hours, uncomplexed nintedanib was separated by filtering the solution with 0.45 µm polyvinylidene fluoride (PVDF) syringe filter. Filtered solutions were quantified for nintedanib using UPLC analysis.(See Supplementary Materials).
2.3 Continuous Variation Method (Job’s Plot Analysis)
Continuous variation method has been used by the researchers for calculation of stoichiometry of the pediatric oncology chemical reactions. Here in the present study, we performed Job’s plot analysis to confirm the stoichiometry of nintedanib and CD during complex formation as per earlier reported methods (Upadhye et al., 2010).
2.4 Preparation of Solid Nintedanib-CD Complex:Solid nintedanib-CD inclusion complex was prepared by freeze drying method as reported earlier (Zhang et al., 2013).
2.5 Characterization of Solid Nintedanib-CD Inclusion Complex: Inclusion complex was characterized by different techniques to confirm the formation of inclusion complex including Fourier Transform Infrared (FT-IR) Spectroscopy, Proton (1H) Nuclear Magnetic Resonance (NMR) Spectroscopy, Differential Scanning Calorimetry (DSC), and X-ray Diffraction (XRD).(See Supplementary Materials).
2.6 In-silico Molecular Modeling Studies:Molecular modeling studies were carried out on a Dell Precision work station with Intel (R) Xeon (R) CPU E51620 v3 @3.50GHz processor. Structure building, docking and analysis were carried out using Accelrys Discovery Studio, GOLD (Genetic Optimization for Ligand Design) suite v5.3 protein ligand docking package and the PyMol molecular graphics software (The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC).
Structure Preparation: The 3D structure of β-CD co-crystalized with α-amylase (PDB: 1JL8) was downloaded from the Protein Data Bank. β-CD was then extracted out of the α-amylase enzyme using GOLD software (Jones, Willett, & Glen, 1995). Since crystal structures of other CDs were not available, they were built in Accelrys Discovery Studio 4.1 visualization software (Discovery Studio Visualizer 4.1; Accelrys, Inc.: San Diego, CA). Geometry of the added functional groups on the β-CD was optimized using Dreiding like force field. All CDs were saved in mol2 format. 2D structures of all CDs are shown in Supplementary Fig. 1.
There are 21 total hydroxyl groups on CD over seven sugar molecules, including 7 primary hydroxyls on the C-6 and 16 secondary hydroxyls, 7 each on C-2 and C-3 of each sugar molecule.Based on alkyl substitution on the CDs (Bansal et al., 1998) the CDs shown in Supplementary Fig. 1 were used for docking. SBE7-β-CD having seven sulfobutyl groups showed highest drug loading capacity (Vangara et al., 2014). Based on their regional selectivity and reactivity between C-6 and C-2,C-3, 4 isomers were built in the order their probability of alkylation (Isomer 1 through 4). Each sugar molecule is restricted to only one Sulfobutyl group to avoid any steric hindrance. 5 hydroxy propyl groups were placed on CD (HP5CD) as the cavasol® comes with a degree of hydroxypropyl substitution from 4.1 to 5.1.
Nintedanib: Nintedanib structure was built in ChemBioDraw Ultra v13.0.2.3021. The energy minimization was done using MM2 force field.Docking: Nintedanib was docked onto various CDs to explore its conformational space within the CD binding pocket using GOLD program. Default GOLD settings were used with 100 GA runs and GOLD score as a scoring function. Binding site was defined using a centroid point for each CD. GOLD score gives information about how good the ligand pose in the binding pocket based on various factors including H-bonding energy, van der waals energy, metal interaction and ligand torsion strain. Best binding pose for each ligand-CD interaction was identified through docking scores and visual inspection considering possible H-bonding interactions.
2.7 Stability Studies of Plain Drug and CD Complex in Different Simulated Physiological Fluids
Stability studies of plain drug and drug CD inclusion complex was done in different simulated bodily fluids at different pH conditions including simulated gastric fluid (SGF) (pH 2), simulated intestinal fluid (SIF) (pH 6.5) and phosphate buffered saline (PBS) (pH 7.4) at 37ºC. Simulated fluids were prepared according to published literature (Marques, Loebenberg, & Almukainzi,2011). Briefly, plain drug or complex were dissolved in methanol or water, respectively and were diluted with simulated fluids to make final concentration of 100 µg/mL. Drug solution was kept in the incubator at 37°C with continuous stirring and aliquots were withdrawn at different time intervals to measure remaining drug in the solution.
2.8 In-vitro Permeability Assay
EpiIntestinal tissue model (SMI196, MatTek Corporation, Ashland, MA) was used to study intestinal permeability of nintedanib as per the supplier’s protocol. Briefly, the plate containing epiIntestinal tissue samples was equilibrated overnight with media supplied with package (SMI100-MM) in a humidified 37°C, 5% CO2 incubator. Following overnight incubation, media was aspirated from both sides and was replenished with 75 µL of A-side transport buffer (1.98 g/L glucose in 10 mM HEPES, HBSS pH 6.5) to the top (apical) chamber, and 200 µL of B-side transport buffer (1.98 g/L HEPES, HBSS pH 7.4) in bottom (basal) chamber. Plain nintedanib (free base or EHS salt) or nintedanib-CD complex (1 µM equivalent) was added to the top or bottom chamber according to experimental design, i.e., to the top if Apical tobasolateral (Ato B) or to the bottom if basolateral to apical (B to A). Samples were collected from the respective receiver chambers after 2 hours and amount of nintedanib was measured using LC-MS/MS method (See Supplementary Material for method details). Permeability coefficient was calculated using
following equation:papp = × where dQ/dt is the flux, C0 is the initial concentration of nintedanib and A is the area of the monolayer.
2.9 In-vitro Bio-Activity Assays
2.9.1 In-Vitro Cell Proliferation Assay: WI-38 cells (2×103 cells/well) were seeded in the 96-well tissue culture plates and after overnight attachment, were serum-starved for 24 hours. After starvation, cells were treated with different growth factors,i.e., PDGF-BB (10 ng/mL) and FGF-β (10 ng/mL), in the presence and absence of nintedanib and nintedanib-CD complex for 72 hours (Cells were treated with nintedaniband nintedanib-CD for 30 min prior to the treatment of growth factors). After 72 hours incubation, cell proliferation was determined using DNA based assay,CyQUANTTM NF cell proliferation assay kit (Thermo Fisher).
2.9.2 In-Vitro Cell Migration Assay: After reaching confluency, WI-38 cells, seeded in 24-well plates, were starved for 24 hours in serum-free media. After starvation, scratches were made in each well using a sterile p200 microtip. Cell debris were washed with media and PBS (pH 7.4) to detach loosely attached cells from the edges, after which the cells were stimulated with PDGF-BB (20 ng/mL) in the presence and absence of nintedanib and nintedanib-CD (1 µM equivalent). Migration of cells from edges was monitored at different time intervals up to 48 hours, and images were taken using a phase contrast microscope (Motic, British Columbia, Canada).
2.9.3 Effects on Collagen Production (Sirius Red Staining and Spectrophotometric Method):The effect of nintedanib-CD complex treatment on the collagen production was measured using Sirius red fast green collagen estimation kit (Chondrex Inc., Redmond, WA). Briefly, WI-38 cells seeded in 24-well plates were serum-starved for 24 hours and subsequently stimulated with TGFβ (5 ng/mL) in the presence and absence of nintedanib and nintedanib CD complex (1 µM equivalent) for further 48 hours in incubator (370C, 5% CO2). After 48 hours, cells were imaged using light microscope and collagen was extracted and quantified as per method provided with kit (See Supplementary Material for detailed method).
3. Results and Discussion:
3.1 Phase-Solubility Studies:
As mentioned earlier, HP-β-CD and SBE-β-CD are safe derivatives of β-CD and are approved for clinical applications and are the most commonly studied CDs in recent years (Brewster & Loftsson, 2007), we tested nintedanib complexation using these two CDs. Phase-solubility diagrams of nintedanib with HP-β-CD and SBE-β-CD arepresented in Fig. 1A. Solubilization capability of the β-CDs can be quantitated using the data obtained from these phase solubility studies. From the diagrams it is depicted that SBE-β-CD showed AL-type phase solubility, i.e.,solubility of nintedanib increased linearly with increasing concentration of CD (Higuchi & Connors, 1965). The apparent solubility of nintedanib was found to be increased to around 500 µM. The AL-type phase diagram revealed 1:1 stoichiometry between nintedaniband SBE-β-CD during formation of inclusion complexes. This is also supported by the slope value obtained from the linear phase solubility diagram, which was found to be lower than one (0.9499) indicating the formation of 1:1 complexation between the drug molecules and SBE-β-CD. The stability constant (Ks) for SBE-βCD based inclusion complex was found to be 689 M1, which is well in the range (1001000 M1) required for appropriate stability of inclusion complexes and also required to improve oral bioavailability of therapeutics (Devasari et al., 2015; Loftsson et al., 2005; Yang et al., 2009). It can also be observed that at lower Nintedanib: SBE-β-CD molar ratio high amount of nintedanib solubilizes whereas this effect is more prominent for HP-β-CD at higher molar ratio. The inclusion complex with HP-β-CD showed linearity at lower concentration of CD, however phase solubility diagram with higher concentration showed AP-type phase solubility which maybe ascribed to the higher (1:2 or 1:4) stoichiometry between nintedanib and HP-β-CD at higher concentration (Higuchi & Connors, 1965). Although the solubility of nintedanib increased up to 700 µM, this AP-type phase solubility resulted in the requirement of higher amount of HP-β-CD to form stable inclusion complex ultimately leading to the increased bulk of the complex. However, for the formulation of dosage form for oral drug administration this might result in increased size of dosage form leading to various formulation issues associated with designing, packaging and storage along with poor patient compliance. As discussed earlier, our aim to prepare inclusion complex was to form a stable complex utilizing minimum quantity of CDs. Hence, based on the results of phase solubility studies, we chose SBE-β-CD nintedanib inclusion complex for further characterization and evaluation of therapeutic activity and intestinal permeability.
3.2 Job’s Plot:
It is known that an alteration in the spectra (measured as shift in λmax)of drug molecule is observed after inclusion owing to the modified microenvironment of solvent due to complexation. The molar ratio at which maximum shift in the λmax of drug occurs is considered selleck chemicals the stoichiometric ratio.The Job’s plot is represented in Fig. 1B. Results of the study showed maximum peak at 0.5 mole fraction value which indicated 1:1 stoichiometry during the formation of inclusion complex. Similar type of stoichiometry has also been observed from phase solubility diagram. Hence, nintedaniband SBE-β-CD molybdenum cofactor biosynthesis were used in 1:1 ratio for the formation of inclusion complex for further characterization and therapeutic evaluation.
3.4 Characterization of Nintedanib-CD Complex:
3.4.1 FT-IR Analysis:
The FT-IR spectra of nintedanib, SBE-β-CD, physical mixture of nintedanib and SBE-β-CD, and nintedanib-CD complex are presented in Fig. 2-(A). The IR spectrum of nintedanib showed characteristic peaks at about 2945, 2933 (C-H stretching, CH3), 2358, 2341, 1705 (C=O stretch, ester), 1653 (C=O stretch, Amide), 1506, 1442, 1292 (C-N stretch), 1222, 1147 and 1089 cm1 (Fig. 2-(A)-i). The FT-IR spectrum of SBE-β-CD exhibited characteristic peaks at about 3431, 2934 and 1022 cm1 which correspond for O-H stretching, C-H stretching and C-O stretching vibration, respectively (Fig. 2-(A)-ii). The spectrum of physical mixture of nintedanib and SBEβ-CD demonstrated a superposition spectrum of both compounds, however less intense absorption peaks of nintedanib at around 1292, 1222, 1147 and 1089 cm1 demonstrated some interaction between nintedaniband SBE-β-CD during formation of physical mixture (Fig. 2-(A)-iii). The FTIR spectrum of nintedanib-CD complex clearly showed that characteristic peaks of nintedanib are disappeared or some less intense peaks are observed at around 1653, 1506 and 1442 cm1 (Fig. 2(A)-iv). These results clearly depicted that some functional groups of nintedanib are included in the cavity of SBE-β-CD to form molecular complex.
3.4.2 1H NMR:
Fig. 2-(B) shows the 1H NMR chemical shifts of nintedanib, SBE-β-CD, the physical mixture of nintedanib and SBE-β-CD, and the inclusion complex of nintedanib-CD. Most of the proton chemical shifts observed for the drug alone were very similar to those observed for the physical mixture and inclusion complex (<0.01 ppm change). However, larger chemical shifts were observed for H1, H2 and H3 as shown in Fig. 2-(B). Specifically, H1 and H2 exhibited a modest change of 0.21 ppm, while H3 showed a substantial change in chemical shift of 0.38 ppm. These chemical shift changes suggest that this aspect of nintedanib scaffold finds itself within the cavity of SBE-β-CD while the rest of nintedanib exists outside of the SBE-β-CD cavity for the inclusion complex. The presence of nintedanib within SBE-β-CD is further supported by large chemical shifts observed for C1 and C2 (0.46 and 0.05 ppm, respectively) when comparing SBE-β-CD alone to the inclusion complex. Molecular modeling studies, discussed later in the manuscript, have also suggested that piperazine ring of nintedanib trapped in cyclodextrin cavity could form stable inclusion complex.
3.4.3 DSC:
Thermal behavior of pure drug and inclusion complex was investigated by thermogravimetric method (Fig. 3-(A)). The thermogram of nintedanib showed a sharp and intense endotherm peak at 255°C ((A)-i), which corresponds to melting point of the drug. Sharp and intense peaks depicted crystalline characteristic of pure drug. The DSC thermogram of SBE-β-CD showed two peaks, one broad peak at 120°C and other peak at 270°C ((A)-ii). First peaks represents to the removal of water molecules from the cavity whereas later presents to the decomposition of CD. Our results are in accordance with previous report (Devasari et al., 2015). The DSC thermogram of physical mixture of nintedaniband SBE-β-CD showed peaks of both molecules, however less intense peaks of both molecules were observed at 255°C and 270°C, and this might be because of possible interaction during formation of physical mixture ((A)-iii). However, sharp peak of nintedanib is disappeared from the DSC thermogram of nintedanib-CD complex, which clearly depicted the formation of inclusion complex and conversion of crystalline nintedanib to amorphous state after complex formation ((A)-iv). Further, in the DSC thermogram of nintedanib-CD complex an endotherm peak at around 355°C was observed, which might be ascribed to the decomposition of complex. Hence, DCS thermogram not only demonstrated the formation of inclusion complex but also showed that after formation of inclusion complex thermo-stability of nintedanib is
improved.
3.4.4 XRD:
Powder XRD (P-XRD) studies were performed to detect the crystallinity of the pure drug and complexed drug. As shown in Fig. 3-(B), nintedanib exhibited several intense and sharp peaks, which confirm the crystalline nature of nintedanib. However, an XRD spectrum of SBE-β-CD has not shown any sharp peak which showed amorphous nature of SBE-β-CD. Further, XRD spectra of nintedanib-CD inclusion complex showed that there are no sharp peaks corresponding to the nintedanib, which depicted that nintedanib might be incorporated in the cavity of CD during
complexation and changed to the amorphous state during freezing process (Vangara et al., 2014).
3.5 Stability Studies:
For the stability studies, we have also used water soluble salt of nintedanib,nintedanibEHS, which is available commercially by prescription. Results of the stability studies in different biological fluids are shown in Fig. 4. Results of the studies showed that although very soluble, nintedanib EHS is highly unstable in PBS (pH 7.4) and is also significantly unstable in simulated intestinal fluid (SIF). Most of the drug (more than 60%) was degraded in PBS (pH 7.4) (Fig. 4A) whereas more than 10% drug was degraded in SIF after 4 hours of incubation at 37°C (Fig. 4B). Nintedanib free base also degraded in PBS however relatively less as compared to EHS salt. In SIF, we did not find stability issues with nintedanib base whereas in simulated gastric fluid (SGF), both base and EHS salt were found stable (Fig. 4C). When nintedanib-CD complex was incubated with SIF and PBS, it was observed that nintedanib was stable for longer time in both fluids as compared to plain drug (Figs. 4A-C). Hence, it could be depicted that stability of nintedanib improved because of its inclusion in the cavity of CD and CD complex would be a better alternative to water soluble EHS salt for dosage form development. By improving stability of nintedanib in SIF and PBS,bioavailability of drug could also be improved.
3.6 Molecular Modeling:
GOLD scores for nintedanib with various substituted CDs are given in Fig. 5B. Docked images of nintedanib over HP5βCD and SBE7βCD are shown in Fig. 5C and 5D.CDs form a tube like structure where drug molecule is trapped or loaded. As substituents on CD are added, based on the number and size, the length of the tunnel is also increased accordingly. Nintedanib is bound to all CDs through a number of hydrophobic interactions. Having multiple aromatic rings, nintedanib was able to show good binding affinity represented by GOLD scores. There are no H-binding interactions were observed with any of CDs. In comparison, HP5CD showed several unfavorable steric interactions over SBE7CD.In all docking poses with CDs, piperazine ring of the nintedanib was trapped in the CD tunnel which may provide additional metabolic stability to this drug when compared to administering free drug along with its increased solubility. In case of SBE7βCD isomer1, where all 7 sulfobutyl ether groups were on the C-6 primary hydroxyl groups reduced docking scores which maybe due to steric hindrance by these groups. When some of these sulfobutyl ether groups were moved to other side of CD as seen in isomer 2, 3 and 4 docking scores were significantly increased. Overall SBE7βCD may have either better drug loading and release characteristics compared to HP5CD drug complexes. Findings from molecular modeling studies are consistent with phase solubility and other experimental studies.
3.7 Effect of Cyclodextrin Complexation on Nintedanib Transport and Efflux across Intestinal Tissue Model by modulating p-gp Efflux:
The major focus of present study was to assess the feasibility of enhancing nintedanib transport across intestinal epithelium by reducing the efflux. Nintedanib is a known substrate for pglycoprotein (p-gp) mediated efflux which may significantly contribute to its low oral bioavailability of 4-5% (Dallinger et al., 2016). Interestingly, in addition to providing solubility enhancement for pharmaceuticals, β -cyclodextrin is also known for modulating p-gp mediated efflux by inhibiting p-gp ATPase activity (Zhang et al., 2013). To assess the capability of CD complexation on the above mentioned characteristics, permeability studies were conducted on the epiIntestinal tissue model which consists of monolayer of human primary small intestine epithelial cells on the cell culture inserts. Till date, most of the in-vitro transport/permeability studies to predict in-vivo intestinal permeability have been performed using Caco-2 cells or Caco-2/HT-29 co-cultures (Artursson, Palm, & Luthman, 2001; Hilgendorfet al., 2000; Nollevaux et al., 2006). However, several reports have suggested limited in-vitro-in-vivo correlation (Gupta, Doshi, & Mitragotri, 2013). Hence, in our study we measured efficacy of CD complexation in modulating permeability of prepared nintedanibusing epiIntestinal tissue model. This model recapitulates the physiology, 3D tissue architecture, and function of small intestine. For this study, we determined cumulative drug transport (both A to B; and B to A), % of loaded transported. Using the permeability data, apparent permeability constants (Papp) for both apical to basal and basal to apical transport, and efflux ratio were calculated using the equations described in the Methods section. To confirm the role of CD complex on the permeability of nintedanib, we have also included water soluble nintedanib EHS in this study. Results of the study demonstrated cumulative amount of drug transported was significantly higher in basolateral to apical direction for all the groups (nin, nin-EHS, and nin-CD complex) (Fig. 6A). However, nin-EHS demonstrated the most prominent increase in B to A transport. At the sametime, CD complexation improved the A to B transport 3folds, and reducing the B to A transport by 2.5 folds as compared to nin-EHS (Fig. 6A). Total % dose transported over 2 hours also showed similar trends with nin-CD demonstrating approximately 4-fold enhancement of A to B transport and 2-fold reduction of B to A transport (Fig. 6B). Nintedanib EHS showed higher Papp for basal to apical transport (6.3×10-7 cm2/sec) as compared to 3.1×10-7 cm2/sec for nin-CD complex (Fig. 6C), This difference in Papp clearly underlined the efficacy of CD-complex in averting B to A efflux for nintedanib, which is depicted in the efflux ratio calculations which showed an efflux ratio of more than 2 for both freebase and EHS salt, demonstrating active efflux. CD complexation however resulted in reduction of efflux ratio to 0.4,which is 6-8 fold lesser than nin and nin-EHS, thus demonstrating the p-gp modulating capabilities of cyclodextrin (Fig. 6D). Earlier reports have suggested that efflux ratio more than 2 represents p-gp efflux (Zhang et al., 2018; Zheng, Chen, & Benet, 2016). Our results are in accordance with previous report showing nintedanib asp-gp substrate (Luedtke et al., 2018). The formation of CD complex resulted in the increase of Papp for apical to basal transport. Moreover, Papp for basal to apical transport decreased when CD complex was used. Earlier, it has been reported that CD complexation could help in reducing p-gp efflux of drugs. As discussed above, CD being hydrophilic in nature, is not substrate for p-gp. Hence, drugs complexed in the interior of CD could also avoid p-gp efflux. Results of our study clearly showed that CD complexation increased transport of nintedanib across intestinal membrane simultaneously reducing p-gp efflux. Increased permeability of nintedanib maybe ascribed to both enhanced aqueous solubility as well as inhibition of p-gp activity by SBE-β-CD (Zhang et al., 2011).However, further mechanistic studies are warranted to assess the effect of CD complexation on the transport mechanism and metabolic profile of nintedanib.
3.8 In-Vitro Bio-Activity
After preparing drug-CD inclusion complex, we tested the bio-activity of inclusion complex in terms of anti-proliferative activity, effect on cells migration, and effect on the synthesis of collagen in growth factors stimulated lung fibroblast cells. As nintedanib is FDA-approved for idiopathic pulmonary fibrosis (IPF), we chose different growth factors which are reported to be involved in the pathogenesis and progression of IPF, including PDGF-BB, bFGF, and TGF-β (Chaudhary et al., 2007; Coward et al., 2010; Richeldi et al., 2014). The activity of CD complex was compared with plain nintedanib.
3.8.1 Anti-Proliferative Activity:
As reported earlier that growth factors stimulated fibroblast proliferation by activating growth factor receptors. Nintedanib has been reported to reduce the growth of activated fibroblasts by blocking growth factor induced proliferation (Wollin, Maillet, Quesniaux, Holweg, & Ryffel, 2014). Results showed that PDGF-BB (Fig. 7A) and bFGF (Fig. 7B) stimulated the proliferation of fibroblast cells, which was reduced by the pre-incubation (30 min) with nintedanib and nintedanib-CD in dose-dependent manner. Interestingly, nintedanib-CD showed significantly higher anti-proliferative activity when compared to plain nintedanib at each tested concentration.
3.8.2 Effects on Migration of Fibroblast Cells:
The effect of nintedanib on the migration of fibroblast cells was evaluated by scratch assay (wound healing). Fig. 8 shows representative images of wound created by scratching serum starved cells with a sterile p200 microtip, following treatment with different compounds for 48 hours. Results of the study depicted that incubation of cells with PDGF-BB stimulated the migration of cells (complete closure of the wound after 48 hours) whereas incubation of cells with PDGF-BB in the presence of nintedaniband nintedanib-CD reduced/blocked the migration of cells. Nintedanib-CD was found to be more potent than nintedanib that maybe correlated to better stability of the drug in CD inclusion complex.
3.8.3 Effect of Treatment on Collagen Deposition:
To further confirm the effect on fibrogenesis, we investigated the effect of nintedanib and nintedanib-CD inclusion complex on the growth factor stimulated collagen deposition in the fibroblast cells. It has already been reported that PDGF-BB, bFGF and VEGF have not shown any effect on stimulation of collagen deposition in fibroblast cells, whereas TGF-β stimulated cells demonstrate collagen deposition significantly higher than normal cells (Hostettler et al., 2014). Hence, in our studies we used TGF-β to stimulate collagen deposition. Results of the study demonstrated that TGFβ stimulated collagen deposition by 127% as compared to control cells (cells without any treatment) (Fig. 9A). Further, it has also been found that pre-treatment (30 min) of cells with nintedanib or nintedanib-CD complex significantly suppressed the stimulated deposition of collagen in the fibroblast cells (Figs. 9A&B). Microscopic observation further confirmed the effect of nintedaniband nintedanib-CD on the collagen deposition. As shown in the Fig. 9B, fibers stained with red color (collagen deposition) are increased in TGF-β stimulated cells whereas cells treated with nintedaniband nintedanib-CD showed significantly less red color fibers thus confirming the reduction of collagen deposition by the treatment.
4. Conclusion:
The present study was aimed at increasing nintedanib’s stability in intestinal environment and improving its intestinal permeability and thus bioavailability. The nintedanib-CD complex was prepared using SBE-β-CD, based on phase solubility studies and molecular modeling results, at 1:1 molar ratio using freeze drying method. Different spectroscopic techniques were utilized to confirm the formation of complex. Formation of the nintedanib-CD complex resulted in improvement of nintedanib stability in PBS and SIF. Further, CD complexation also marginally increased the bioactivity of nintedanib in terms of cell proliferation, collagen deposition, and cell migration. Transport studies using 3D epiIntestinal model have suggested a significant increase in transport of nintedanib across intestinal epithelium by reducing the p-gp efflux. Results of the studies outline a premise to use nintedanib-CD complex for development of oral dosage forms with increased bioavailability.