Intestine-on-a-Chip Microfluidic Model for Efficient in Vitro Screening

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Intestine-on-a-chip Microfluidic Model for Efficient in vitro Screening of Oral Chemotherapeutic Uptake Kyall Pocock, Ludivine Delon, Vaskor Bala, Shasha Rao, Craig Priest, Clive A. Prestidge, and Benjamin Thierry ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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ACS Biomaterials Science & Engineering

Intestine-on-a-chip microfluidic model for efficient in vitro screening of oral chemotherapeutic uptake Kyall Pocock1, Ludivine Delon1, Vaskor Bala2, Shasha Rao2, Craig Priest1, Clive Prestidge2,3 and Benjamin Thierry1,3*

1

Future Industries Institute, University of South Australia, Mawson Lakes Campus, Mawson

Lakes, Adelaide, South Australia 5095, Australia 2

School of Pharmacy and Medical Sciences, University of South Australia, City East Campus,

Adelaide, South Australia 5000, Australia 3

ARC Centre of Excellence in Convergent Bio and Nano Science and Technology, University of

South Australia, South Australia 5095, Australia

KEYWORDS: Intestine-on-a-chip, 7-ethyl-10-hydroxycamptothecin (SN38), prodrugs, biomicrofluidic, intestinal drug transport.

ACS Paragon Plus Environment

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ABSTRACT Many highly effective chemotherapeutic agents can only be administered intravenously as their oral delivery is compromised by low gastro-intestinal solubility and permeability. SN-38 (7ethyl-10-hydroxycamptothecin) is one such drug; however recently synthesized lipophilic prodrugs offer a potential solution to the low oral bioavailability issue. Here we introduce a microfluidic based intestine-on-a-chip (IOAC) model, which has the potential to provide new insight into the structure-permeability relationship for lipophilic prodrugs. More specifically, the IOAC model utilizes external mechanical cues that induce specific differentiation of an epithelial cell monolayer to provide a barrier function that exhibits an undulating morphology with microvilli expression on the cell surface; this is more biologically relevant than conventional Caco-2 Transwell models. IOAC permeability data for SN38 modified with fatty acid esters of different chain length and at different molecular positions correlate excellently with water-lipid partitioning data and has the potential to significantly advance their pre-clinical development. In addition to advancing mechanistic insight into the permeability of many challenging drug candidates, we envisage the IOAC model to also be applicable to nanoparticle and biological entities.


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INTRODUCTION The reliance on animal models is a major obstacle in the development of efficient formulations for the oral delivery of novel therapeutics including biologics.1 In addition to the inherent ethical issues associated with the use of such animal models, they often fail to accurately predict the response of human tissues to orally administered drugs. Animal models also provide only minimal mechanistic information and are time and resource intensive.2 Alternatively, in the standard in vitro Transwell model, epithelial cell monolayers grown on porous substrates are used to model the intestinal epithelium and study drug transport in vitro under static fluid conditions.3-4 This model is time and labor intensive as it requires approximately 3 weeks to form a fully differentiated and confluent monolayer. The Transwell model does not accurately mimic the human intestinal physiological environment as it lacks dynamic fluid flow applying shear stress to cells during culture and does not provide a continual source of nutrients.5-7 Owing to the limitation of these in vitro and in vivo models in mimicking complex human physiology and disease conditions, the rate of successful preclinical to clinical cancer trial translation is on average below 8%.8 Therefore, there has been an increased interest in developing alternative in vitro models that better recapitulate the complex functions of the human digestive track. The integration of microfluidics with living biological systems has paved the way for the exciting “organ-on-a-chip” concept, which aims at developing advanced in vitro models that replicate the key features of human tissues/organs. In pioneering work, Imura et al. demonstrated the feasibility of culturing human epithelial Caco-2 cells inside a microfluidic device to produce a cellular monolayer and then evaluating drug intestinal absorption.9 A more advanced model was subsequently developed based on the culture of Caco-2 cells under physiologically relevant conditions including peristalsis-like motions and liquid flow as well as using 3D scaffolds.10-11


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When cultured in such mimetic conditions, Caco-2 cells spontaneously underwent morphogenesis of 3D intestinal villi features characteristic of human intestinal epithelium, including the presence of tight junctions and mucus. Importantly, these were not present in conventional Caco-2 cell Transwell model.12 In a human intestine, microvilli are actin-based membrane protrusions that facilitate a number of biological processes, including the absorption and transepithelial transport of biomolecules through the intestinal epithelium. The pivotal role of fluid shear stress in the formation of microvilli by epithelial cells grown in a microfluidic environment was recently demonstrated.13 Despite these exciting developments, the application of such “intestine-on-a-chip” (IOAC) models in the pharmaceutical field are s still in its infancy. Of particular interest is the ability to better predict the transport of bioactive agents through the intestinal epithelium. We report here on the design and characterization of a microfluidic IOAC model towards evaluating the transport of various prodrugs of the anticancer drug SN38 (7-ethyl-10-hydroxy camptothecin). SN38 is a semisynthetic analogue of camptothecin and is one of the most potent compounds within the camptothecin family which inhibits DNA Topoisomerase I blocking the DNA regulation of topoisomerase cleavage complexes.14 Being a Biopharmaceutical Classification System (BCS) Class-IV drug with low water solubility and low transmucosal permeability,15-16 it is only available clinically in its water soluble prodrug form (irinotecan) and is delivered via intravenous infusion for advanced colon cancers as monotherapy or combination therapy.17 However, the use of irinotecan is limited by the low and erratic conversion to active SN38 in the body (2-8%), short half-life and severe dose limiting diarrhea and neutropenia.18 We have recently designed a range of lipophilic SN38 prodrugs aimed at improving its lipophilicity, stability, and enhancing its transmucosal permeability towards the ultimate objective of enabling


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oral SN38-based chemotherapy.19 We demonstrate the feasibility of using an IOAC microfluidic model to evaluate the transepithelial transport of these lipophilic prodrugs.

EXPERIMENTAL SECTION Microfabrication The intestine-on-a-chip model is a four layered microdevice comprising 3 layers of polydimethylsiloxane (PDMS) (Sylgard 184, USA) and a polycarbonate (PC) membrane with a thickness of 15 µm and a pore size of 3µm (Millipore, USA). As can be seen in Figure 1, layer 1 contains the basal chamber and channels and layer 2 is the PC membrane. Layer 3 contains the apical chamber and channels with 3 mm wells that align with the inlets in layer 1 and the inlets for layer 3 itself that act as bubble traps. Layer 4 seals the microdevice and contains 1.5 mm holes that align with all bubble traps and are the four connection ports for the inlet and outlet tubing. The master molds were fabricated using standard soft lithography. In short, SU-8 50 (MicroChem, USA) was spun to produce layers 150 µm in thickness on a Karl Suss Delta 80 spin coater (Suss MicroTec, Germany) on silicon wafers, then chamber and channel design was written via a Dilase 650 mask writer (Kloe, France), followed by development and thermal hardening of the SU-8. PDMS (10:1, w/w) was cast on to the silicon micropatterned master mold and cured for 2 hours at 70 °C. Incursions were made and the PDMS molds removed. Layer 3 has two 3 mm holes (bubble traps) punched and two 1.5 mm holes, which are then oxygen plasma treated and bound to layer 4 that is a flat 3 mm thick slab of PDMS. Through the existing holes in layer 3, layer 4 has four 1.5 mm holes punched (order specific to ensure the holes align)


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to act as ports for connection of input and output tubing’s. Layer 1 requires no holes to be punched.

Microdevice Functionalization and Sealing A custom build pulse plasma polymerization unit described elsewhere was utilized for bonding PC membrane and PDMS surfaces.20 Allyl glycidyl ether (AGE) monomer was coated on desired bonding surfaces using an input power of 25 W with a duty cycle of 1 ms/20 ms where the initial monomer pressure was 0.2 mbar, the pressure prior to introducing the monomer was set at 1 x 10-3 mbar. AGE monomer was coated on the surfaces for 60 s before the duty cycle was engaged for 120 s resulting in a reactive epoxy functionalized surface. Both sides of the PC membrane were functionalized before being immersed in 0.1 % poly-L-lysine solution (Sigma Aldrich, Australia) for 2 hours and dried under nitrogen resulting in a primary amine available for bonding with the reactive epoxy functionalized surface of the PDMS. The layers were aligned, pressed together to initiate the conjugation reaction and left overnight at 70 °C for the bond to strengthen.

Microdevice Cell Culture Human intestinal epithelial Caco-2 cells (ATCC) were cultured in 75 mm2 tissue culture flasks in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma Aldrich) supplemented with 10 % fetal bovine serum (Sigma Aldrich), 1 % L-glutamine (Thermo Fisher Scientific, Australia) and 1 % streptomycin/ penicillin (Thermo Fisher Scientific). Caco-2 cells were maintained at 37 °C and at


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5 % CO2 levels in a humidifying incubator. Prior to use the cells were grown to 70-80 % confluence before seeding inside a microdevice. All cells used for experimental work were between passages 20-40. For comparison, Transwell models were seeded 2 x 105 cells/cm2 with a media change every 2-3 days. IOAC microdevices were first sterilized using UV treatment for 1 hour on both sides of the PC membrane. Gas impermeable Tygon tubing (Cole Palmer, USA) was connected to the inlet and outlet ports followed by flushing the device with 70 % ethanol using a peristaltic pump (Langer Instruments, USA) for 30 minutes before infusion of phosphate buffer solution (PBS) (Sigma Aldrich) for 20 minutes followed by DMEM for 1 hour. Matrigel (Invitrogen, USA) was used as an extracellular matrix. 0.3 % v/v Matrigel in DMEM, injected using a peristaltic pump at 2 µL/min and was incubated for 2 hours under static conditions to form a coating on the apical side of the PC membrane. Next, Caco-2 cells were manually injected in the apical channel of the device at a density of 3 x 105 cells/cm2 and left to adhere to the Matrigel treated PC for at least 4 hours. The cell media flow rate post attachment for the top chamber was set at 0.4 µL/min, equivalent to an averaged 0.02 dyne cm-2 of fluid shear stress in the culture chamber (the applied shear stress was calculated using an adapted Poiseuille flow and microfluidic wall shear stress equation as developed by Zhang et al.).21-22 24 hours post cell attachment, the basal chamber flow was initiated and DMEM was infused at 0.4 µL/min. The Caco-2 cells were grown for 5 days under these fluidic conditions to form a confluent monolayer using a closed loop system, containing approximately 5 mL of cell media (configuration can be seen in the supporting information, Figure S1). The cell number was monitored daily using bright field microscopy on an inverted Eclipse Ti-E Nikon microscope (Nikon, Japan) with the monolayer being confirmed


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by immunostaining using calcein (Sigma Aldrich) for live cell membrane staining and Hoechst (Sigma Aldrich) for live nuclei staining.

Immunostaining Cell tight junctions were used to validate the growth of an intact cell monolayer. Anti- occludin tight junction immunostaining was performed as recommended by the manufacturer (catalogue number ab31721, Abcam, UK); Caco-2 cell monolayer inside the IOAC microdevice was washed with cold PBS for 15 minutes. Cells were fixed by injecting 4 % paraformaldehyde in PBS (Thermo Fisher Scientific) at 2 µL/min continuously for 15 minutes followed by a 10 minute PBS wash. 0.5 % Triton X-100 v/v (ChemSupply) was perfused for 15 minutes at 2 µL/min for cell permeabilisation with a 5 minute PBS wash. 3 % bovine serum albumin (Sigma Aldrich) solution was prepared in PBS and used as a blocking agent and incubated at room temperature for 1 hour. A dilute rabbit anti-occludin 2 µg/ µl solution (Abcam) was injected in the IOAC apical chamber and incubated for 2 hours at room temperature. PBS was injected for 10 min to wash out any unreacted primary antibody and replaced by a 1 µg/ µl goat anti-rabbit IgG (AlexaFluor® 488) (Abcam) in 1 % BSA, which was incubated at room temperature for 1 hour. A final 10 minute PBS wash was perfused before the IOAC model was carefully deconstructed, the membrane cut in to sections and mounted for immunofluorescent imaging using a LMS 710 with Elyra confocal microscope (Zeiss, Germany). All washing and staining steps were carried out at a flow rate of 10 µL/min.

Scanning Electron Microscopy Imaging


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To obtain electron microscopy images of the cellular monolayer, the IOAC device was deconstructed and the PC membrane with the cell monolayer grown on the surface was isolated. Cells were fixed using an alternate method than previously mentioned, using 4% paraformaldehyde for 15 minutes and 2.5 % glutaraldehyde (Sigma Aldrich) for 2 hours followed by serial dehydration using 25, 50, 75, 95 and 100 % ethanol in water. Samples were transferred and chemically dried using hexamethyldisilazane (HDMS) (Sigma Aldrich), initially in a 1: 2 solution of HDMS: ethanol for 20 minutes, repeated for a further 20 minutes at 2: 1 and finally 100 % HDMS for 20 minutes before replacing this solution and leaving overnight to evaporate. The sample was sputter coated with ~20 nm of gold with an Edwards TF500 sputter coater (HHV, UK) and imaged using a Merlin with GEMINI II scanning electron microscope (Zeiss, Germany).

Transport of SN38 Prodrug through the IOAC Epithelial Layer SN38 and the prodrugs SN38-undecanoate C20, SN38-undecanoate C10 and SN38-propionate C20 were synthesized as previously described.19 SN38 and prodrug solutions were injected and recirculated in closed loop in the apical device at 10 mM. 30 µL was collected at regular 30 minute intervals from the apical and basal outlets. The concentration of the SN38 drug and prodrugs were determined using a Shimadzu Prominence ultrafast liquid chromatography (UPLC) system (Shimadzu, Japan) connected to a CBM-20A communications bus module, LC-20AD liquid chromatography pump, SIL-20A HT autosampler and a photodiode array (PDA) detector. An isocratic flow rate was used at 1.2 ml/min for separation in a reverse phase Phenomenex Kinetex C18 (150 × 4.6 mm; 5 µm)


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analytical column. The mobile phase had the pH adjusted to 3 and comprised of a 50:50 mixture of 25 mM NaH2PO4 and acetonitrile. Stock solutions of SN38 and the 3 prodrugs were prepared at 10 mg/ml in DMSO and sonicated for 10 minutes. Standard curves ranging from 0.1 to 10 µg/ml (R2 > 0.99) were constructed. Samples recovered from the apical and basal sides of the membrane were diluted with the mobile phase 10 and 2 fold respectively. Maximum absorbance was recorded for SN38, SN38-undecanoate C20, SN38-propioanoate C20 at λ = 380 nm while SN38-undecanoate C10 was recorded at λ = 360 nm.

Data Analysis The raw data as measured using UPLC was used to calculate the apparent permeability (Papp) which is defined as the amount of analytes transported across the cell monolayer per unit time.23 This was calculated using Equation 1.

= × ×

(1)

Where, dQ/dt is the rate of drug transport from the apical to basolateral chamber, A is the exposed cell area (0.18096 cm2) and Co is the initial drug concentration in the apical chamber.

Statistical Analysis All data presented is expressed as mean ± standard deviation. Statistical analysis was performed using a one-way analysis of variance (ANOVA) and any multiple data sets were compared using


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a student T-test. Any difference between groups with p < 0.05 were considered statistically significant.

RESULTS AND DISCUSSION

Figure 1. Intestine-on-a-chip microdevice design. (A) Schematic of human intestinal location and structure. (B) Deconstructed 3D schematic of the 4 layers IOAC device. A plasma polymerization process is used to bond the various layers and seal the device. The PC membrane (2) separates the apical chamber (3) from the basal chamber (1). The basal and apical chambers are perfused independently through a peristatic pump. The top layer (4) contains the bubble traps and seals the device. (C) Schematic of Caco-2 cell monolayers grown on PC membranes under dynamic and static fluid flow conditions.

Design and characterization of the intestine-on-a-chip microdevice.


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The IOAC device design (Figure 1) consists of 4 layers in total, 3 layers are made of PDMS and 1 layer made of a thin PC membrane. Two chambers perfusable with independent fluidic lines are separated by the PC membrane and form apical and basal chambers analogous to the structure commonly found in the standard in vitro Transwell model. Both perfusion chambers provide nutrient delivery in the form of cell culture media to cells during the culture period. In the IOAC design, larger inlet ports are punched and sealed with a top layer of PDMS with smaller diameter inlets to create on-chip bubble traps. Efficient elimination of bubbles is essential for long term perfusion cell culture due to intrinsic difficulties associated with microfluidics and the potential for spontaneous bubble nucleation in cell culture media.24-25 The PC membrane used here allows for a direct comparison with Transwell models that utilize the same material for cell attachment and culture.26 Caco-2 cells were selected as the epithelial cell of choice to use in the model as they are the most widely used cell line for mimicking the human gastrointestinal epithelial barrier.27-28 After functionalization of the apical side of the PC membrane with Matrigel, Caco-2 cells were injected inside the apical section of the IOAC device and left to attach for 4 hours. At this time point, the apical chamber was then perfused with DMEM cell culture media supplemented with FBS at a flow rate calculated to induce an averaged fluid shear stress of 0.02 dyne cm-2. In agreement with previous studies,29-32 dense Caco-2 cell monolayers were obtained typically after 5 days of culture in these dynamic culture conditions. The cell monolayers on the PC membrane were prepared for imaging with confocal microscopy which confirmed that the IOAC model allows for the formation of cell monolayers that possess undulating 3D morphology as shown by F-actin mapping of the apical surface of cell monolayer. This feature somewhat replicates the morphology of the native intestinal epithelium (Figure 2). On the other hand, Caco-2 cells grown


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inside the IOAC microdevice in static conditions presented a flatter morphology as shown by the 3D confocal image reconstruction of F-actin (Figure 2A). This finding is in agreement with previous studies showing that in static fluidic conditions, such as those used in Transwell models, the monolayer surface is flat and lacks some of the key critical components, such as 3D morphology and microvilli like structures, that exist within the human intestinal epithelium.33-34

Figure 2. 3-dimensional confocal reconstruction and orthogonal views of cell monolayer. Confocal images of Caco-2 cell monolayers grown within the IOAC microdevice under (A) static and (B) dynamic fluidic conditions. F-actin is stained using phalloidin (green) and cell nuclei stained using 4',6-diamidino-2-phenylindole (DAPI) (blue).

Next, immunofluorescence observation was used to further characterize the monolayer cultured inside the IOAC device under dynamic growth conditions at day 5. Imaging of occludin protein expression (Figure 3A and 3B) confirmed that intact epithelial-like monolayers are formed inside the IOAC within 5 days of dynamic culture. Occludin expression is characteristic of the epithelial barrier function and is essential to enable valid permeability studies. The constant delivery of cell media to growing cells, coupled with the application of a constant shear stress


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has been shown to accelerate growth and differentiation compared to the standard Transwell static culture model that requires 21 days to achieve full differentiation.35-37 The application of the shear stress induces faster differentiation and promotes the formation of a confluent monolayer within 5 days. The dynamic conditions encountered in the IOAC microdevice replicate more closely those that exist within the human intestinal tract which may account for these differences.12

Figure 3. Immunofluorescent confocal images of tight junction protein occludin (green) in a Caco-2 cell monolayer grown under static fluidic conditions in a Transwell (A) and under dynamic fluidic conditions in a IOAC microdevice (B).

Under higher magnification inspection of the Caco-2 cell monolayers formed inside the IOAC microdevice with scanning electron microscopy, microvilli-like subcellular structures after 5 days of culture were observed for monolayer grown under dynamic conditions (Figure 4B, 4C


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and 4D). On the other hand, cells grown under static conditions in a Transwell model and fixed showed a lack of microvilli after 5 days (Figure 4A). To confirm the SEM observations, antivillin staining was performed on both static and dynamic fluidic models. Villin is an actinbinding protein found in microvilli epithelium. Villin expression was higher in IOAC monolayers than in Transwell culture (supporting information). While microvilli formation can be achieved in the Caco-2 cell Transwell model, it generally requires 21 days and a mechanical shear, chemical or electrostatic stimulation.38-40 Microvilli are a key component of epithelial cells and are actin-based membrane features that aid in a range of epithelial functions, including secretion, absorption and mechanotransduction.41-43. In addition, microvilli provide a significant increase in surface area which can lead to a higher interaction and uptake of a compound of interest.44 Microvilli formation can be initiated by a number of protein based factors but not until recently had the effects of external factors such as fluid shear stress been investigated.13 In agreement with Miura et al., the presence of mechanical shear associated to the dynamic flow culture condition in the IOAC model allowed for the formation of well-defined microvilli within 5 days. Combined these results confirmed the suitability of the proposed IOAC microdevice for the preparation of epithelial-like monolayers that mimic human small intestinal microenvironment. In this design, the apical perfusion chamber containing the cellular monolayer can be perfused with the compound of interest. Collection of media from the basal channel allows for the determination of transport through the monolayer and therefore, calculation of permeability coefficients.


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Figure 4. Scanning electron micrographs. Image A, SEM micrograph of Caco-2 cells grown to confluency on PC membrane under static flow conditions (Transwell). Images B, C and D SEM micrographs of Caco-2 cell monolayer in IOAC device under dynamic conditions.

Validation of the IOAC microdevice for permeability coefficient studies


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To validate the suitability of the cellular monolayer formed in the IOAC microdevice for determining the transport of compounds via either paracellular or transcellular pathway, two model compounds with well-established transmucosal permeability were selected; caffeine and atenolol with high and low permeability, respectively.45-46 Permeability coefficients of 34.5 ± 10.3 10-4 cm/sec for caffeine and 3.8 ± 1.8 10-4 cm/sec for atenolol were determined. These permeability coefficients are significantly higher than those typically obtained using Caco-2 monolayers grown in the standard Transwell model as well as those obtained using rat mucosal tissues mounted within an Ussing chamber (Table 1). On the other hand, the coefficients obtained using the IOAC device are within one order of magnitude to those reported for human tissue in vivo (2.93 ± 0.16 10-4 cm/sec for caffeine and 0.02 ± 0.02 10-4 for atenolol). The relative value of the ratio of permeability coefficients for these two model molecules (i.e. ratio of Papp for atenolol and caffeine) was also in good agreement with standard Transwell results (broad range), rat mucosal tissues mounted in Ussing chamber determined permeability’s (9 vs 14.4) as well as measurements obtained for human intestinal epithelium (9 vs 14.6).47 The difference between the magnitudes of the permeability coefficients might be attributed to the higher surface area associated to the 3D-like morphology of the cell monolayer in the IOAC microdevice under dynamic conditions as well as the highly functional nature of these cells. Based on these considerations, IOAC models may be anticipated to provide a better mimic of the intestinal epithelium in comparison to static monolayers grown in Transwell models. IOAC models also possess significant advantages over Ussing chamber models based on animal tissue that are both labor and resource intensive.48 It is also noteworthy that direct kinetic comparisons of transport variables such as permeability coefficients under complex human intestinal in vivo environments are rare.49-50 Technique mimicking intestinal perfusions, such as IOAC microfluidic models,


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have the potential to enable direct in vitro investigation of single parameters such as intestinal permeability, absorption, biliary secretion and efflux; without the complex influences of metabolism, pH changes, dissolution and intestinal mobility that are experienced in vivo.51-53 It is well established that the physicochemical properties of drugs such as molecular weight, logP, logD, polar surface area and hydrogen bonding, are directly related to their overall intestinal absorption potential.54-55 Suggesting microfluidic IOAC models have a significant role to play in the future to study the intestinal permeability of newly developed drugs and biomolecules other than those in the existing well-characterized chemical space, such as lipophilic conjugated prodrugs, as well as to better define the relationship between physiochemical properties and intestinal permeability.56-57

Table 1. In vitro permeability of caffeine and atenolol through IOAC device, a modular Ussing Chamber system, Transwell model and human in vivo (mean ± SD, n = 3).

Papp Ussing Chamber – rat tissue (cm/sec)19

Range of Papp Transwell model (cm/sec)58-60

Papp Human in vivo (cm/sec)61-63

34.5 ± 10.3 x 10-4

0.19 ± 0.001x10-4

0.85 ± 0.01x10-4 to 0.26 ± 0.015x10-4

2.93 ± 0.16 x 10-4

3.8 ± 1.80 x 10-4

0.0136 ± 0.002x10-4

0.001 ± 0.002x10-4 to 0.004 ± 0.01x10-4

0.2 ± 0.02 x 10-4

Papp IOAC device Compound (cm/sec)

caffeine

atenolol


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Permeability Coefficient of Lipophilic SN38 Prodrugs Towards the development of oral SN38-based chemotherapy with efficient oral absorption, we have recently developed novel lipophilic SN38 prodrugs. Alkyl ester prodrugs of SN38 at positions C10 or C20 were synthesized as described previously.19 In brief, the saturated fatty acids undecanoic acid and propionic acids were conjugated to SN38 at the C20 position to synthesize the SN38-undecanoate C20 and SN38-propionoate C20 respectively. Undecanoic acid was also conjugated at the C10 position. The SN38-undecanoate C20 possesses favorable lipophilicity (i.e., logP >5, Table 2), as well as high stability in simulated gastrointestinal conditions, making it an excellent candidate for oral delivery. This lipophilic prodrug as well as the parent compound SN38 were perfused through the IOAC device, and the transport from the apical chamber to the basal chamber was determined using UPLC and used to calculate permeability coefficients Papp. No significant absorption of SN38 or its prodrugs occurred in the PDMS based IOAC system.


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Table 2. SN38 and prodrug structures, calculated logP (ACD ChemSketch) and prodrug solubility in soybean oil (mean ± SD, n = 3). (adapted from Bala et al.19)

Structure Compound Name

logP

Lipid Solubility (mM)

R1

R2

SN38

H

H

2.31 ± 0.78

0.0078 ± 0.00082

SN38-undecanoate C20

-

CH3(CH2)9CO

7.66 ± 0.72

0.61 ± 0.15

SN38-undecanoate C10

CH3(CH2)9CO

-

6.89 ± 0.66

1.53 ± 0.0095

SN38-propionoate C20

-

CH3CH2CO

3.41 ± 0.72

0.11 ± 0.035


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Figure 5. In vitro permeability coefficients for SN38 and SN38-undecanoate C20 as measured using the IOAC device and rat intestinal mucosal membrane mounted in an Ussing Chamber. *p < 0.05 (mean ± SD, n = 3) vs SN38.

A 2.9 fold increase in the transport of the lipophilic SN38-undecanoate C20 through the IOAC was measured in comparison to SN38 as can be seen in Figure 5 (Papp = 6.65 ± 0.75 10-4 cm/sec vs. 2.31 ± 0.19 10-4 cm/sec respectively). These results can be qualitatively compared with data obtained using rat intestinal mucosal tissues mounted in Ussing chambers. Permeability coefficients of 3.6× 10-6 cm/sec for SN38 and 7.5 × 10-6 cm/sec for SN38-undecanoate C20 were obtained.19 In both models, the lipophilic prodrug was transported more efficiently to the basal chamber than the unmodified SN38 (x2.9 in IOAC vs x 2.1 in Ussing chamber). However, in


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agreement with the data obtained in the validation study, the permeability coefficients were approximately two orders of magnitude higher for the IOAC device than for the Ussing chamber system with rat tissue (10-6 vs 10-4). As noted above, the absolute values obtained with the IOAC device might be more relevant to that of the human gut, but this point warrants further studies. Current literature reports extensively on rat in vivo data but intestinal permeability studies in other relevant mammals such as dogs or pigs are rare, although more relevant dosages can be accommodated in these models.64 In addition, only very limited data is available in human. Altogether, this provides a strong impetus for the further development of IOAC models able to better predict the permeability of drugs and biomolecules through the human intestinal epithelium.


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Papp 9

6.9

6.7

8

7 6

5.3

6

5

3.4

4 3

10

3

2.3

4

logP

(10-4) (cm/sec)

logP

7.7

8

Papp IOAC device

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.3

2

2

1 0

0

38 N S

20 10 C C te te t a a a n o o io an an p c c o de de pr n n -u -u 38 8 8 N 3 3 S SN SN 20 C e

Figure 6. In vitro permeability coefficients of poorly soluble SN38 and prodrugs through an IOAC device compared to calculated logP demonstrating the linked relationship between the permeability and logP for SN38 and prodrugs (mean ± SD, n = 3).

Next, two additional lipophilic SN38 prodrugs with various levels of solubility were tested in the IOAC model to demonstrate the feasibility of correlating the physicochemical characteristics of such formulation with transport. The modulation of a drug solubility via encapsulation or conjugation with lipophilic moieties is an effective approach to enhancing transport across cellular barriers.65 Figure 6 shows Papp values obtained using the IOAC device as well as the


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logP values for the tested prodrugs. The highest permeability was achieved for the SN38undecanote C20 at 6.7 ± 0.75 10-4 cm/sec. Derivatization with undecanoate at the C10 position rather than the C20 resulted in a slight reduction in the solubility of the prodrug which translated into reduction of the permeability coefficient (5.3 ± 0.24 10-4 cm/sec). Similarly, derivatization at the C20 position with a shorter alkyl chain yielded a significant decrease (p< 0.05 vs C20 prodrugs) in the prodrug logP which translated to a significant decrease in the permeability coefficient (3 ± 0.27 10-4 cm/sec). Along with other physicochemical properties such as molecular weight and charges, the lipophilicity of a drug directly influence its ability to permeate through a cellular monolayer. Within the range of interest in oral formulation, increased lipophilicity enhances transport through Caco-2 monolayer grown in the Transwell model.66 The observed correlation between the solubility’s of the SN38 prodrugs and their permeability coefficients further demonstrate the relevance of the IOAC model. Note that further studies should investigate the presence of mucus and its role in these IOAC permeability coefficients as poorly soluble drugs can interact with mucus glycoprotein which would in turn influence their intestinal transport. It is of significant importance to precisely elucidate the rate-limiting step(s) involved in drug uptake following oral administration as many of the mechanisms have not yet been fully established. Predictions of human intestinal permeability is commonly based on a number of parallel processes, such as carrier-mediated absorption, passive transcellular diffusion and carrier mediated efflux; which all differ along sections of the intestinal tract.67-68 On the other hand, it is widely accepted that the intestinal epithelium mediates the transport rate for both high and low permeability compounds in vivo, regardless of the overall transport mechanism. This further strengthens our findings as here we have shown a relationship between the gastrointestinal


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solubility of a range of lipophilic prodrugs and the permeability in human intestinal epithelial cells cultured under dynamic flow conditions. However complex questions such as those regarding the dissolution, chemical and enzymatic stabilities remain unanswered. Nevertheless IOAC models’ ability to provide realistic platforms for testing orally administered chemotherapeutic drug candidates have the potential to aid in the formulation of next generation of advanced oral formulations.

CONCLUSION In agreement with previous reports, the microfluidic IOAC model described here allowed for the preparation within a 5 day period of Caco-2 cell monolayers that possess 3D undulating villi-like surfaces, and exhibit tight junctions and microvilli. This model was utilized to determine the permeability coefficients of lipophilic prodrugs of SN38. As expected, strong correlation was obtained between the prodrug logP and the experimentally determined permeability coefficients. However, the magnitudes of the IOAC permeability coefficients was significantly higher than those measured using the standard Ussing chamber set up with rat tissues. A similar observation was made for the two reference molecules used in the validation phase, caffeine and atenolol. These findings suggest that IOAC microdevices mimicking the key characteristics of the intestinal epithelium have a significant potential to improve on the in vitro current assays used to screen oral formulations. Further studies are warranted to demonstrate the validity of permeability coefficients determined using such IOAC microdevices as well as to fully elucidate the links between the structure and phenotype of cellular monolayers formed under dynamic


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culture conditions and epithelial permeability. The presence of mucus and its potential impact on IOAC permeability coefficients should also be investigated.

ADDITIONAL CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Figures S1 –S3: Schematic of perfusion system experimental set-up used for IOAC microdevices and further confocal data characterizing the cell monolayer, including 3D reconstructions and orthogonal projections.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding This work is supported by the Australian Research Council Linkage Grant LP150100032.


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Notes The authors declare no competing financial interest. Acknowledgement This work was performed in part at the South Australian node of the Australian National Fabrication Facility under the National Collaborative Research Infrastructure Strategy to provide nano and microfabrication facilities for Australia’s researchers.

REFERENCES 1. Hooijmans, C.; Geessink, F.; Ritskes-Hoitinga, M.; Scheffer, G., A Systematic Review of the Modifying Effect of Anaesthetic Drugs on Metastasis in Animal Models for Cancer. Plos One 2016, 11 (5). 2. Hogenesch, H.; Nikitin, A. Y., Challenges in pre-clinical testing of anti-cancer drugs in cell culture and in animal models. J Control Release 2012, 164 (2), 183-186. 3. Kis, O.; Zastre, J.; Hoque, M.; Walmsley, S.; Bendayan, R., Role of Drug Efflux and Uptake Transporters in Atazanavir Intestinal Permeability and Drug-Drug Interactions. Pharm. Res. 2013, 30 (4), 1050-1064. 4. Willenberg, I.; Von Elsner, L.; Steinberg, P.; Schebb, N., Development of an online-SPELC-MS method for the investigation of the intestinal absorption of 2-amino-1-methyl-6phenylimidazo [4,5-b]pyridine (PHIP) and its bacterial metabolite PHIP-M1 in a Caco-2 Transwell system. Food Chem. 2015, 166, 537-543. 5. Amandalkauffman; Chrissaferguson, Alternative Functional In Vitro Models of Human Intestinal Epithelia. Frontiers in Pharmacology 2013, 4. 6. Abdayem, R.; Callejon, S.; Portes, P.; Kirilov, P.; Demarne, F.; Pirot, F.; Jannin, V.; Haftek, M., Modulation of transepithelial electric resistance ( TEER ) in reconstructed human epidermis by excipients known to permeate intestinal tight junctions. Exp. Dermatol. 2015, 24 (9), 686-691. 7. Mukherjee, T.; Squillantea, E.; Gillespieb, M.; Shao, J., Transepithelial Electrical Resistance is Not a Reliable Measurement of the Caco-2 Monolayer Integrity in Transwell. Drug Delivery, 2004, Vol.11(1), p.11-18 2004, 11 (1), 11-18.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 41

8. Mak, I.; Evaniew, N.; Ghert, M., Lost in translation: animal models and clinical trials in cancer treatment. American Journal Of Translational Research 2014, 6 (2), 114-118. 9. IMURA, Y.; ASANO, Y.; SATO, K.; YOSHIMURA, E., A Microfluidic System to Evaluate Intestinal Absorption. Analytical Sciences 2009, 25 (12), 1403-1407. 10. Kim, H. J.; Huh, D.; Hamilton, G.; Ingber, D. E., Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab on a chip 2012, 12 (12), 2165. 11. Chen, Y.; Lin, Y.; Davis, K.; Wang, Q.; Rnjak-Kovacina, J.; Li, C.; Isberg, R.; Kumamoto, C.; Mecsas, J.; Kaplan, D., Robust bioengineered 3D functional human intestinal epithelium. Scientific Reports 2015, 5. 12. Kim, H. J.; Ingber, D. E., Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation. Integrative Biology 2013, 5 (9), 1130-1140. 13. Miura, S.; Sato, K.; Kato-Negishi, M.; Teshima, T.; Takeuchi, S., Fluid shear triggers microvilli formation via mechanosensitive activation of TRPV6. Nature Communications 2015, 6, 8871. 14. Vangara, K.; Ali, H.; Lu, D.; Liu, J.; Kolluru, S.; Palakurthi, S., SN-38-Cyclodextrin Complexation and Its Influence on the Solubility, Stability, and In Vitro Anticancer Activity Against Ovarian Cancer. AAPS PharmSciTech 2014, 15 (2), 472-482. 15. Bansal, T.; Mishra, G.; Jaggi, M.; Khar, R. K.; Talegaonkar, S., Effect of P-glycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats. European Journal of Pharmaceutical Sciences 2009, 36 (4), 580-590. 16. Bala, V.; Rao, S.; Boyd, B. J.; Prestidge, C. A., Prodrug and nanomedicine approaches for the delivery of the camptothecin analogue SN38. Journal of Controlled Release 2013, 172 (1), 48-61. 17. Bala, V.; Rao, S.; Prestidge, C. A., Facilitating gastrointestinal solubilisation and enhanced oral absorption of SN38 using a molecularly complexed silica-lipid hybrid delivery system. European Journal of Pharmaceutics and Biopharmaceutics 2016, 105, 32-39. 18. Santi, D. V.; Schneider, E. L.; Ashley, G. W., Macromolecular prodrug that provides the irinotecan (CPT-11) active-metabolite SN-38 with ultralong half-life, low C(max), and low glucuronide formation. Journal of medicinal chemistry 2014, 57 (6), 2303. 19. Bala, V.; Rao, S.; Li, P.; Wang, S.; Prestidge, C. A., Lipophilic Prodrugs of SN38: Synthesis and in Vitro Characterization toward Oral Chemotherapy. Molecular Pharmaceutics 2016, 13 (1), 287-294. 20. Thierry, B.; Griesser, H. J.; Prestidge, C. A.; Palms, D.; Kurkuri, M.; Al-Ejeh, F.; Brown, M. P.; Shi, J. Y., Plasma functionalized PDMS microfluidic chips : towards point-of-care capture of circulating tumor cells. Journal of materials chemistry 2011, 21 (24), 8841-8848.


28

Page 29 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


21. Zhang, X.; Huk, D. J.; Wang, Q.; Lincoln, J.; Zhao, Y., A microfluidic shear device that accommodates parallel high and low stress zones within the same culturing chamber. Biomicrofluidics 2014, 8 (5). 22. Lentle, R.; Janssen, P., Physical characteristics of digesta and their influence on flow and mixing in the mammalian intestine: a review. Journal of Comparative Physiology B 2008, 178 (6), 673-690. 23. Hubatsch, I.; Ragnarsson, E. G. E.; Artursson, P., Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nature Protocols 2007, 2 (9), 2111. 24. Stucki, J. D.; Guenat, O. T., A microfluidic bubble trap and oscillator. Lab on a Chip 2015, 15 (23), 4393-4397. 25. Wang, Y.; Lee, D.; Zhang, L.; Jeon, H.; Mendoza-Elias, J.; Harvat, T.; Hassan, S.; Zhou, A.; Eddington, D.; Oberholzer, J., Systematic prevention of bubble formation and accumulation for long-term culture of pancreatic islet cells in microfluidic device. Biomed. Microdevices 2012, 14 (2), 419-426. 26. Bhattacharjee, S.; Opstal, E.; Alink, G.; Marcelis, A.; Zuilhof, H.; Rietjens, I., Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells. J. Nanopart. Res. 2013, 15 (6), 1-14. 27. Gamboa, J. M.; Leong, K. W., In vitroandin vivomodels for the study of oral delivery of nanoparticles. Adv Drug Deliv Rev 2013, 65 (6), 800-810. 28. Artursson, P.; Palm, K.; Luthman, K., Caco-2 monolayers in experimental and theoretical predictions of drug transport. Advanced Drug Delivery Reviews 2012, 64, 280-289. 29. Gargam, N.; Darrasse, L.; Raynaud, J. S.; Ginefri, J. C.; Robert, P.; Poirier‐Quinot, M., Experimental system to detect a labeled cell monolayer in a microfluidic environment. Journal of Magnetic Resonance Imaging 2015, 42 (4), 1100-1105. 30. Kim, S.; Lee, J.; Choi, I.; Kim, Y.-C.; Lee, J.; Sung, J., A Microfluidic Device with 3-D Hydrogel Villi Scaffold to Simulate Intestinal Absorption. Journal of Nanoscience and Nanotechnology 2013, 13 (11), 7220-7228. 31. Huang, C.; Ramadan, Q.; Wacker, J. B.; Tekin, H. C.; Ruffert, C.; Vergres, G.; Silacci, P.; Gijs, M. A. M., Microfluidic chip for monitoring Ca 2+ transport through a confluent layer of intestinal cells. RSC Advances 2014, 4 (95), 52887-52891. 32. Chi, M.; Yi, B.; Oh, S.; Park, D.-J.; Sung, J.; Park, S., A microfluidic cell culture device (µFCCD) to culture epithelial cells with physiological and morphological properties that mimic those of the human intestine. Biomedical Microdevices 2015, 17 (3), 1-10. 33. Shen, C.; Meng, Q.; Zhang, G., Design of 3d printed insert for hanging culture of caco-2 cells. Biofabrication 2015, 7 (1), 015003.


29


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Page 30 of 41

34. Shah, M.; Madan, P.; Lin, S., Elucidation of intestinal absorption mechanism of carvedilol-loaded solid lipid nanoparticles using Caco-2 cell line as an in-vitro model. Pharmaceutical Development And Technology 2015, 20 (7), 877-885. 35. Mehling, M.; Tay, S., Microfluidic cell culture. Current Opinion in Biotechnology 2014, 25, 95-102. 36. Raimes, W.; Rubi, M.; Super, A.; Marques, M. P. C.; Veraitch, F.; Szita, N., Transfection in perfused microfluidic cell culture devices: A case study. Process Biochemistry 2016. 37. Tazawa, H.; Sato, K.; Tsutiya, A.; Tokeshi, M.; Ohtani-Kaneko, R., A microfluidic cell culture system for monitoring of sequential changes in endothelial cells after heat stress. Thrombosis Research 2015, 136 (2), 328-334. 38. Takahashi, J.; Ogihara, K.; Naya, Y.; Kimura, F.; Itoh, M.; Iwama, Y.; Matsumoto, Y.; Toshima, G.; Hata, K., An in vitro assay system for antihyperlipidemic agents by evaluating lipoprotein profiles from human intestinal epithelium-like cells. 3 Biotech 2013, 3 (3), 213-218. 39. Vandrangi, P.; Lo, D. D.; Kozaka, R.; Ozaki, N.; Carvajal, N.; Rodgers, V. G. J., Electrostatic properties of confluent Caco-2 cell layer correlates to their microvilli growth and determines underlying transcellular flow. Biotechnology and bioengineering 2013, 110 (10), 2742. 40. Papafragkou, E.; Hewitt, J.; Park, G.; Greening, G.; Vinjé, J., Challenges of Culturing Human Norovirus in Three-Dimensional Organoid Intestinal Cell Culture Models. PLoS One 2013, 8 (6). 41. Bailey, D.; Turnbull, G.; Bartsch, R.; Begent, R.; Sapsford, R.; Ciclitira, P. J., Comparative Analysis of Adult and Fetal Human Small Intestinal Microvilli. Digestion 58 (2), 155-160. 42. Vandrangi, P.; Lo, D.; Kozaka, R.; Ozaki, N.; Carvajal, N.; Rodgers, V., Electrostatic Properties of Confluent Caco-2 Cell Layer Correlates to Their Microvilli Growth and Determines Underlying Transcellular Flow. Biotechnol. Bioeng. 2013, 110 (10), 2742-2748. 43. Grant, C. N.; Mojica, S. G.; Sala, F. G.; Hill, J. R.; Levin, D. E.; Speer, A. L.; Barthel, E. R.; Shimada, H.; Zachos, N. C.; Grikscheit, T. C., Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function. American journal of physiology. Gastrointestinal and liver physiology 2015, 308 (8), G664. 44. Vandrangi, P.; Lo, D. D.; Kozaka, R.; Ozaki, N.; Carvajal, N.; Rodgers, V. G. J., Electrostatic properties of confluent Caco‐2 cell layer correlates to their microvilli growth and determines underlying transcellular flow. Biotechnology and Bioengineering 2013, 110 (10), 2742-2748. 45. Bansal, T.; Singh, M.; Mishra, G.; Talegaonkar, S.; Khar, R. K.; Jaggi, M.; Mukherjee, R., Concurrent determination of topotecan and model permeability markers (atenolol, antipyrine, propranolol and furosemide) by reversed phase liquid chromatography: Utility in Caco-2 intestinal absorption studies. J. Chromatogr. B 2007, 859 (2), 261-266.


30

Page 31 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


46. Kuwayama, K.; Inoue, H.; Kanamori, T.; Tsujikawa, K.; Miyaguchi, H.; Iwata, Y.; Miyauchi, S.; Kamo, N.; Kishi, T., Interactions between 3,4-methylenedioxymethamphetamine, methamphetamine, ketamine, and caffeine in human intestinal Caco-2 cells and in oral administration to rats. Forensic Sci. Int. 2007, 170 (2-3), 183-188. 47. Da Silva, L. C.; Da Silva, T. L.; Antunes, A. H.; Rezende, K. R., A Sensitive MediumThroughput Method to Predict Intestinal Absorption in Humans Using Rat Intestinal Tissue Segments. Journal of Pharmaceutical Sciences 2015, 104 (9), 2807-2812. 48. Alqahtani, S.; Mohamed, L.; Kaddoumi, A., Experimental models for predicting drug absorption and metabolism. Expert Opinion On Drug Metabolism & Toxicology 2013, 9 (10), 1241-1254. 49. Sun, D.; Lennernas, H.; Welage, L.; Barnett, J.; Landowski, C.; Foster, D.; Fleisher, D.; Lee, K.-D.; Amidon, G., Comparison of Human Duodenum and Caco-2 Gene Expression Profiles for 12,000 Gene Sequences Tags and Correlation with Permeability of 26 Drugs. Pharmaceutical Research 2002, 19 (10), 1400-1416. 50. Cook, J., A Technique to Estimate In Vivo Dissolution Profiles Without Data from a Solution. The AAPS Journal 2012, 14 (3), 433-436. 51. Tannergren, C.; Engman, H.; Knutson, L.; Hedeland, M.; Bondesson, U.; Lennernäs, H., St John's Wort Decreases the Bioavailability of R ‐ and S ‐verapamil Through Induction of the First‐pass Metabolism. Clinical Pharmacology & Therapeutics 2004, 75 (4), 298-309. 52. Wu, B.; Kulkarni, K.; Basu, S.; Zhang, S.; Hu, M., First-Pass Metabolism via UDPGlucuronosyltransferase: a Barrier to Oral Bioavailability of Phenolics. Journal of Pharmaceutical Sciences 2011, 100 (9), 3655-3681. 53. Higashino, H.; Hasegawa, T.; Yamamoto, M.; Matsui, R.; Masaoka, Y.; Kataoka, M.; Sakuma, S.; Yamashita, S., In vitro-in vivo correlation of the effect of supersaturation on the intestinal absorption of BCS Class 2 drugs. Molecular pharmaceutics 2014, 11 (3), 746. 54. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews 2012, 64, 4-17. 55. Davies, M. N.; Bayry, J.; Tchilian, E. Z.; Vani, J.; Shaila, M. S.; Forbes, E. K.; Draper, S. J.; Beverley, P. C. L.; Tough, D. F.; Flower, D. R.; Diemert, D. J., Toward the Discovery of Vaccine Adjuvants: Coupling In Silico Screening and In Vitro Analysis of Antagonist Binding to Human and Mouse CCR4 Receptors (Finding Adjuvants In Silico). PLoS ONE 2009, 4 (11), e8084. 56. Skold, C.; Winiwarter, S.; Wernevik, J.; Bergstrom, F.; Engstrom, L.; Allen, R.; Box, K.; Comer, J.; Mole, J.; Hallberg, A.; Lennernas, H.; Lundstedt, T.; Ungell, A.; Karlen, A., Presentation of a structurally diverse and commercially available drug data set for correlation and benchmarking studies. Journal Of Medicinal Chemistry 2006, 49 (23), 6660-6671.


31


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Page 32 of 41

57. Persson, A.; Pettersson, C.; Rosén, J., Multivariate Data Analysis of Factors Affecting the In Vitro Dissolution Rate and the Apparent Solubility for a Model Basic Drug Substance in Aqueous Media. Pharmaceutical Research 2010, 27 (7), 1309-17. 58. Da Silva, L. C.; Da Silva, T. L.; Antunes, A. H.; Rezende, K. R., A Sensitive Medium‐ Throughput Method to Predict Intestinal Absorption in Humans Using Rat Intestinal Tissue Segments. J Pharm Sci 2015, 104 (9), 2807-2812. 59. Hellinger, É.; Veszelka, S.; Tóth, A. E.; Walter, F.; Kittel, Á.; Bakk, M. L.; Tihanyi, K.; Háda, V.; Nakagawa, S.; Dinh Ha Duy, T.; Niwa, M.; Deli, M. A.; Vastag, M., Comparison of brain capillary endothelial cell-based and epithelial (MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate blood–brain barrier penetration models. Eur J Pharm Biopharm 2012, 82 (2), 340-351. 60. Volpe, D.; Asafu-Adjaye, E.; Ellison, C.; Doddapaneni, S.; Uppoor, R.; Khan, M., Effect of Ethanol on Opioid Drug Permeability Through Caco-2 Cell Monolayers. The AAPS Journal 2008, 10 (2), 360-362. 61. Li, H.; Jin, H.-E.; Shim, W.-S.; Shim, C.-K., An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system. Drug Development and Industrial Pharmacy, 2013, Vol.39(10), p.1515-1522 2013, 39 (10), 1515-1522. 62. Ruan, L.-P.; Chen, S.; Yu, B.-Y.; Zhu, D.-N.; Cordell, G. A.; Qiu, S. X., Prediction of human absorption of natural compounds by the non-everted rat intestinal sac model. European Journal of Medicinal Chemistry 2006, 41 (5), 605-610. 63. Lennernäs, H., Animal data: The contributions of the Ussing Chamber and perfusion systems to predicting human oral drug delivery in vivo. Advanced Drug Delivery Reviews 2007, 59 (11), 1103-1120. 64. Carlert, S.; Akesson, P.; Jerndal, G.; Lindfors, L.; Lennernas, H.; Abrahamsson, B., In Vivo Dog Intestinal Precipitation of Mebendazole: A Basic BCS Class II Drug. Molecular Pharmaceutics 2012, 9 (10), 2903-2911. 65. Yasmin, R.; Rao, S.; Bremmell, K. E.; Prestidge, C. A., Porous Silica-Supported Solid Lipid Particles for Enhanced Solubilization of Poorly Soluble Drugs. AAPS Journal 2016, 18 (4), 876-885. 66. Camenisch, G.; Alsenz, J.; van de Waterbeemd, H.; Folkers, G., Estimation of permeability by passive diffusion through Caco-2 cell monolayers using the drugs' lipophilicity and molecular weight. European Journal of Pharmaceutical Sciences 1998, 6 (4), 313-319. 67. Amidon, G.; Lennernäs, H.; Shah, V.; Crison, J., A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability. Pharmaceutical Research 1995, 12 (3), 413-420. 68. Charkoftaki, G.; Dokoumetzidis, A.; Valsami, G.; Macheras, P., Elucidating the Role of Dose in the Biopharmaceutics Classification of Drugs: The Concepts of Critical Dose, Effective


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In Vivo Solubility, and Dose-Dependent BCS. Pharmaceutical Research 2012, 29 (11), 31883198.


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For Table of Contents Use Only

Intestine-on-a-chip microfluidic model for efficient in vitro screening of oral chemotherapeutic uptake Kyall Pocock, Ludivine Delon, Vaskor Bala, Shasha Rao, Craig Priest, Clive Prestidge and Benjamin Thierry


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Figure 1. Intestine-on-a-chip microdevice design. (A) Schematic of human intestinal location and structure. (B) Deconstructed 3D schematic of the 4 layers IOAC device. A plasma polymerization process is used to bond the various layers and seal the device. The PC membrane (2) separates the apical chamber (3) from the basal chamber (1). The basal and apical chambers are perfused independently through a peristatic pump. The top layer (4) contains the bubble traps and seals the device. (C) Schematic of Caco-2 cell monolayers grown on PC membranes under dynamic and static fluid flow conditions. 392x171mm (96 x 96 DPI)



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Figure 2. 3-dimensional confocal reconstruction and orthogonal views of cell monolayer. Confocal images of Caco-2 cell monolayers grown within the IOAC microdevice under (A) static and (B) dynamic fluidic conditions. F-actin is stained using phalloidin (green) and cell nuclei stained using 4',6-diamidino-2phenylindole (DAPI) (blue). 166x52mm (149 x 149 DPI)


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Figure 3. Immunofluorescent confocal images of tight junction protein occluding (green) in a Caco-2 cell monolayer grown under static fluidic conditions in a Transwell (A) and under dynamic fluidic conditions in a IOAC microdevice (B). 171x83mm (96 x 96 DPI)



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Figure 4. Scanning electron micrographs. Image A, SEM micrograph of Caco-2 cells grown to confluency on PC membrane under static flow conditions (Transwell). Images B, C and D SEM micrographs of Caco-2 cell monolayer in IOAC device under dynamic conditions. 234x201mm (96 x 96 DPI)


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Figure 5. In vitro permeability coefficients for SN38 and SN38-undecanoate C20 as measured using the IOAC device and rat intestinal mucosal membrane mounted in an Ussing Chamber. *p < 0.05 (mean ± SD, n = 3) vs SN38. 166x114mm (167 x 167 DPI)



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Figure 6. In vitro permeability coefficients of poorly soluble SN38 and prodrugs through an IOAC device compared to calculated logP demonstrating the linked relationship between the permeability and logP for SN38 and prodrugs (mean ± SD, n = 3). 165x133mm (150 x 150 DPI)


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TOC 354x206mm (96 x 96 DPI)


Intestine-on-a-Chip Microfluidic Model for Efficient in Vitro Screening

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