A Novel, Ready-to-Use System for In Vitro Intestinal
Contents
1. DADMEcell caco apy A Novel Ready to Use System for In Vitro Intestinal Absorption Evaluations Manufactured and Distributed Under License From Dui Jee oe i advanced in vitro cell technologies CACOREADY TECHNICAL NOTE TABLE OF CONTENTS INTRODUCTION ccc cca weed ode eee ok KEY FEATURES AND BENEFITS TECHNICAL DESCRIPTION 2 0005 Kit CONTOMES 255 sce tiea royi ei a es Applications seeen aod te eo eek eB Wid ee Assay TYP Skeno a nn huta a EER o eR SS DOSCHPUON 26 33 peng eee hd eRe wR ED RRE BIOSAFETY i208 cere inoue iy ei diene bee SHIPPING HANDLING amp UNPACKING THE CACOREADY KIT ere cies te eee de eee SIPPING edaria nihet a peared he be EN Unpacking 2 hii oe kok ee REE OS PRS Changing the Shipping Medium 44 RESISTANCE OF THE CACOREADY SYSTEM TO COMMON SOLUBILIZERS SOLVENTS STABILITY OF CACOREADY DURING SHIPMENT amp STORAGE 00 0 000 e Stability of CacoReady During Shipment Stability of CacoReady After Receipt REFERENCES oiia icone ee dee tee eed n toa ea ee OTHER CACOREADY PRODUCTS CaCO GODlCE 3 och ede gie erir ki ERRE WWANSPOMtPlUS 33 6258 steak 3 Paget henii Gt 2 INTRODUCTION The CacoReady system is a new ready to use concept for n vitro intestinal absorption evaluation It is an integrated tool designed to minimize time and costs for preclinical screening of oral c2. 77 236 3235 4053 Harlan Street 311 Tel 510 428 9600 2009 ADMEcell Inc ADMEcell and the ADMEcell logo are trademarks and or registered trademarks of ADMEcell Inc Emeryville CA 94608 Fax 510 428 9666 www admecell com info admecell com
3. brane permeability and sol ubility in the intestinal lumen As many compounds entering development have high lipophilicity solubi lizers are often used to enhance the bioavailability of drugs having poor aqueous solubility Accordingly the potential of solvents to disrupt the barrier status is one of the largest factors limiting wider use of the Caco 2 system The study presented below focuses on the effect of different solubilizers on the barrier status of the CacoReady system performed to determine the predictability of the model for the oral absorption of poorly water soluble drugs in humans Lucifer yellow paracellular permeability values obtained in presence of various solubilizing excipients and sol vents such as polyethylenglycol Solvent 1 up to 5 propyleneglycol Solvent 2 up to 10 dimethyl sul foxide DMSO Solvent 3 acetonitrile Solvent 4 and methanol Solvent 5 up to 2 recommended 0 5 CACOREADY TECHNICAL NOTE were compared to those obtained using only a HBSS control Figure 4 The permeability values show that the CacoReady 21 day barrier remains stable after exposure to the solvents The results support the use of the CacoReady system for poorly water soluble drugs to predict their intestinal absorption in humans T Solvent 2 Solvent 3 Solvent4 Solvent 5 T HBSS Solvent 1 FIGURE 4 Lucifer yellow paracellular flux through the Caco R
4. eady barrier in the presence of different solvents after 1 hour solvent exposure STABILITY OF CACOREADY DURING SHIPMENT amp STORAGE Stability of CacoReady During Shipment The functional stability of CacoReady as a ready to use product during shipping and storage is presented below Thirteen day Caco 2 cell monolayers were maintained in shipping medium for 1 3 4 5 and 7 days and barrier status was evaluated by TEER measurement before applying and 72 hours after removing the ship ping medium The CacoReady barrier status showed no reduction in barrier properties until 5 days in ship ping medium This indicates that CacoReady can be stored and transported at room temperature up to 4 days without loss of Caco 2 barrier properties 6000 E Before E After 40004 30004 20004 1000 0 1 day 3 days i 4 days 5 days 7 days 50004 TEER ohms cm2 FIGURE 5 Stability of CacoReady in shipping medium during shipping and storage 5 CACOREADY TECHNICAL NOTE Stability of CacoReady After Receipt One of the greatest benefits of the CacoReady Sys tem is its ready to use and user friendly aspect The stability of the kit i e how long after delivery the plates can be stored before barrier properties are lost is presented below Immobilization was maintained for 72 hours at room temperature The shipping medium was removed and TEER Tran
5. ents The exclusive and proprietary Ensures the stability of the shipping media cell barrier properties CacoReady kits can be used Provides flexibility in up to 9 days after delivery designing and planning experimental work TABLE 1 Features and benefits of CacoReady system TECHNICAL DESCRIPTION Kit Contents HTS Transwell 24 or 96 plates e Technical sheet brief description of the kit quality control instructions for use Applications CacoReady is intended for n vitro evaluation of Oral absorption efficiency e Compound screening e Oral bioavailability e Oral toxicity e Mechanisms for oral intestinal absorption e Research on new delivery systems Assay Types CacoReady can be used to perform the following assays e Permeability assays for recommended volumes please consult the product User Manual e Immunocytochemistry e Protein extraction Description The CacoReady kit consists of differentiated Caco 2 cell barriers shipped on day 13 of differentiation and provides 14 day polarized cultures of Caco 2 cells on polycarbonate microporous filters in 24 or 96 well plates Figure 1 Apical compartment Caco 2 monolayer Basal compartment Microporous filter FIGURE 1 Representation of polarized Caco 2 monolayer in Tran swell system CACOREADY TECHNICAL NOTE The innovative CacoReady shipping medium main tains the properti
6. es and integrity of the cell barrier providing users with a robust screening tool that can be used up to 9 days after its been received Figure 2 shows confocal analysis of occludin staining revealing tight junctions status in the CacoReady cell barrier FIGURE 2 Confocal analysis of occludin staining green revealing tight junctions status in the CacoReady cell barrier Figure 3 shows the apparent permeability coefficient of standard compounds characterization 8E 03 7E 03 5 6E 03 E 5e 034 E 4E 03 5 g 3E 03 5 a 2E 03 1E 03 5 E OE 03 mimm mim m M ma 3 Bi Dig Biosa 2 2 Z Ot p o a0 2h ME D a a E ere Bp S a EE gt amp 2 i 1 3 D 1 Oo to n 1 4 a1 f 4 o 1 re 1 1 2 1 1 Q 1 1 1 F Oh ob O i gee ok j ka ES o ie 4 ot a ia ae i 4 t Oy Paracellular Transporters passive diffusion Transcellular PGP passive diffusion substrate FIGURE 3 Apparent permeability coefficient of standard com pounds characterization BIOSAFETY For research use only This product contains cell lines from human origin and as with any material produced from human origin it is recommended to observe full safety precautions when handling this material CACOREADY TECHNICAL NOTE The following information is provided with each lot of products e Lot Number e Transepithelial Electrical Resistance Ohm cm2 e Lucifer Yellow Paracellular Flux e Apparen
7. le more than one plate at a time while changing the shipping medium Re solidification of the shipping medium could provoke mechanical damage to the cell monolayers 1 Take one CacoReady plate and one basal plate out of the incubator and place them both in the biosafety cabinet oriented the same direction 2 Fill a sterile container with 50 ml of pre warmed 37 C Caco 2 culture medium 3 Open the CacoReady plate and the basal plate placing the lids next to the plates facing upwards 4 Genily lift up the apical upper section of the Ca coReady plate and place it on the basal plate 5 Remove all the liquefied shipping medium from the basal compartments of the CacoReady plate by aspirating with the manifold or a Pasteur pipette 6 Gently move the apical section of the Caco Ready plate from the basal plate back to the top of the original CacoReady plate 7 Remove all the liquefied shipping medium from the apical compartments of the CacoReady plate by aspirating with the manifold or a Pasteur pipette 8 Cover the CacoReady plate with its lid then put back into the cell culture incubator 9 Repeat steps 1 9 for each plate After the shipping medium has been changed to fresh Caco 2 medium the plates should be kept in the incu bator until Monday RESISTANCE OF THE CACOREADY SYSTEM TO COMMON SOLUBILIZERS SOLVENTS Absorption of orally administrated drugs is mainly determined by their mem
8. o allow the transport medium to completely liquefy 4 While the shipping medium is liquefying prepare everything you will need to replace the shipping medium with fresh Caco 2 cell culture medium e Cell culture biosafety cabinet e Standard Caco 2 cell culture medium pre warmed to 37 C e Aspiration system e Automatic multichannel micropipettor e Standard 24 well plates Costar 3526 for as many as CacoReady plates as you have received e Sterile containers for culture medium 5 Prepare the basal plates for changing the medium a Fill a sterile container with 50 ml of Caco 2 me dium that has been warmed to 37 C b In the biosafety cabinet unwrap one 24 well basal plate Costar 3526 for each CacoReady plate Open the plates and place the lids by the plates facing upwards c Program the automatic multichannel pipette for a 900 ul sequence Aspirate 900 ul of warm 37 C Caco 2 medium from the sterile contain er and dispense 900 ul of this medium into each of the 24 wells of the basal plates d Put the lid on each plate and place the plates in the incubator e Re warm the Caco 2 medium by placing it back in the 37 C bath Note Culture media should be replaced every 48 72 hours Once the shipping medium has been liquefied re place it with fresh Caco 2 culture medium following the steps below Steps 2 10 must be performed using sterile technique in the biosafety cabinet Caution Never hand
9. ompounds CacoReady is perfectly suited for assessing perme ability and can easily be adopted for high throughput screening of compounds CacoReady is an innovative cell based assay with the following features e Provides differentiated Caco 2 barrier 21 day sys tem plated on HTS Transwell 24 plates or HTS Transwell 96 plates e Transwells are integrated to allow easier handling e Patented proprietary shipping medium is stable at room temperature allows up to 5 days of transpor tation storage at room temperature e User friendly system and easy handling e Cost effective for any size laboratory The CacoReady system is designed in collaboration with industrial partners and validated independently by several biotech and pharmaceutical companies KEY FEATURES AND BENEFITS Features Benefits Ready to use kits are stable at Easy handling and storage room temperature Allows end users to avoid in Reduces operative costs house maintenance and han and provides significant dling of Caco 2 cell culture time savings User friendly and suitable for Adaptable to high through automation put automated systems Cost savings both in direct and indirect costs Avoids reagent prepara tion plastic ware and many hours of tedious cell culture work No requirement for cell culture facility equipment logistics Flexibility of the CacoReady Allows testing of poorly system water soluble compon
10. s Epithelial Electrical Resistance was measured after 1 3 5 and 9 days in standard culture conditions No changes were observed in the TEER values throughout 72 hours of immobilization The stability of the CacoReady barrier from 1 to 9 days after im mobilization is displayed below Figure 6 This data demonstrates the flexibility the CacoReady system brings to designing and planning experiments 4000 T 5 3000 o 5 20004 o tH 10004 0 T T T T T Before 1 day 3 days 5 days 9 days reception cell culture day 13 FIGURE 6 Stability of the Caco 2 barrier evaluated by TEER measurement at different days after immobilization in CacoReady shipping medium REFERENCES 1 Le Ferrec et al n vitro Models of the Intestinal Barrier 2001 ATLA 29 649 668 ECVAM Work shop Report 46 Yee S n vitro permeability across Caco 2 cells colonic can predict n vivo small intestinal ab sorption in man fact or myth Pharm Res 1997 Jun 14 6 763 6 Takahashi Y Kondo H Yasuda T Watanabe T Kobayashi S Yokohama S Common solubiliz ers to estimate the Caco 2 transport of poorly water soluble drugs nt J Pharm 2002 Oct 10 246 1 2 85 94 Artursson P Karlsson J Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial Caco 2 cells Biochem Biophys Res Commun 1991 Mar 29 175 3 880 5 Ruba
11. s W et al Flux measurements across Caco 2 monolayers may predict transport in human large intestinal tissue J Pharm Sci 1996 Feb 85 2 165 9 Stewart BH et al Comparison of intestinal per meabilities determined in multiple n vitro and in situ models relationship to absorption in hu mans Pharm Res 1995 May 12 5 693 9 CACOREADY TECHNICAL NOTE OTHER CACOREADY PRODUCTS CacoGoblet Mucus secreting CacoGoblet represents a predictive model for compounds or formulations with a passive diffusion transport pathway Similar to CacoReady this product is also designed as a ready to use kit and has been designed to eliminate in house cell culture work CacoGoblet is offered in 24 and 96 well plates TransportPlus This ready to use kit provides polarized cultures of Caco 2 and or TC7 cell barriers and or MDCK and is designed to allow comparative permeability evalua tion in a single assay with 2 or 3 different cell lines As with CacoReady and CacoGoblet it is designed to eliminate in house cell culture work enabling re searchers to allocate more of their resources to focus on their core research CACOREADY TECHNICAL NOTE ADMEcell and the ADMEcell logo are trademarks of ADMEcell Inc CacoReady is a trademark of Advancell Transwell is a registered trademark of Corning Life Sciences and all other trademarks are the property of their respectve owners 3ADMEcell ADMEcell Inc Toll Free 8
12. t Permeability cm min e Manufacturing Date e Expiration Date SHIPPING HANDLING amp UNPACKING THE CACOREADY KIT Note This is a general overview for handling unpack ing and further processing of CacoReady products For more detailed and comprehensive instructions please refer to the product User Manual Shipping CacoReady is shipped Mondays through Thurs days from the production facility in California Normal delivery time is 1 or 2 days within the continental United States Hawaii Canada and other US territo ries Therefore you should receive the kits between Tuesday and Friday of the week they are shipped You do not need to work with the plates until Friday Unpacking 1 Upon receipt open the outer box and remove the inner box 2 Open the inner box and remove the plastic zip lock bags containing the HTS Transwell 24 plates Leave the zip lock bags open The plastic zip lock bags containing the HTS Tran swell 24 plates should be kept at room tem perature 15 25 C until Friday of the week they are received Note the shipping medium must be changed on Friday of the week the kit is received Changing the Shipping Medium 1 On Friday open the plastic zip lock bags and remove the HTS Transwell 24 plates still at room temperature 2 Unwrap the HTS Transwell 24 plates and care fully pull off the Parafilm 3 Place the HTS Transwell 24 plates in the cell culture incubator for 4 hours t
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