Caco-2 cell assay

We then used this Caco-2 cell assay to categorize representative fluoroquinolone drug substance permeability [50], The drugs demonstrated some concentration-dependent permeability indicative of active drug transport. Based upon comparison to labetalol, ciprofloxacin was classified as a LP drug, whereas levofloxacin, lomefloxacin, and ofloxacin were classified as HP drugs, which matched their human in vivo bioavailabilities. All four fluoroquinolone drugs were subject to efflux transport (ciprofloxacin > lomefloxacin > rhodamine 123 > levofloxacin > ofloxacin). 

Many laboratories use CACO-2 cells as a standard method for assessment of efflux. Since the standard CACO-2 cell assay is very well established, easy to use, reproducible and reliable the corresponding efflux assay can give valuable and helpful data for project support in a screening approach. A prerequisite for the interpretation of efflux data is a characterization of efflux transporters present in the system used and a set of standard efflux markers checked regularly (like digoxin for MDR1). 

Because poor bioavailability represents one of the principal causes of compound failure, absorption and metabolism assays feature prominently in lead optimization screens. As oral administration represents the ideal route of drug delivery, models that predict low absorption across the gastrointestinal (GI) tract are commonly employed. Caco-2 cell assays have been... 

PAMPA-biomimetic-Caco-2-comparison Several in vitro assays have been developed to evaluate the Gl absorption of compounds. Our aim was to compare three of these methods (/) the BAMPA method, which offers a HT, noncellular approach to the measurement of passive transport ( ) the traditional Caco-2 cell assay, the use of which as a HT tool is limited by the long cell differentiation time (21 days) and (// ) The BioCoat HTS Caco-2 assay system, which reduces Caco-2 cell differentiation to three days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations (f= 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat HTS Caco-2 assay system does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21 -day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption. 

For compounds where PAMPA showed much lower permeability than the Caco-2 cell assay, it can be concluded that absorption transport is the primary permeability mechanism for these compounds. 

The chemical structure of crizotinib contains both pyridine and piperidine moieties which have measured p/fa values of 5.4 and 8.9, respectively. The compound thus exhibits pH-dependent solubility 0.034 mg/mL in water, 41 mg/mL in simulated gastric fluid (pH = 1.6), and 0.19 mg/mL in simulated intestinal fluid (pH = 6.5). Crizotinib displayed moderate human hepatocyte clearance and low-to-moderate permeability in Caco-2 cell assays. The compound also displayed good and consistent bioavailability in preclinical species, such as rat, dog, and monkey [%F 63 (rat), 65 (dog), 42 (monkey)] along with a relatively large volume of distribution (Vss about 13 L/kg) and moderate clearance values which translated to a long ti/2 (5.5-17 h). ... 

Yamashita et al. [82] also studied the effect of BSA on transport properties in Caco-2 assays. They observed that the permeability of highly lipophilic molecules could be rate limited by the process of desorption off the cell surface into the receiving solution, due to high membrane retention and very low water solubility. They recommended using serum proteins in the acceptor compartment when lipophilic molecules are assayed (which is a common circumstance in discovery settings). 

Yamashita et al. [82] added up to 10 mM taurocholic acid, cholic acid (cmc 2.5 mM), or sodium laurel sulfate (SLS low ionic strength cmc 8.2 mM) to the donating solutions in Caco-2 assays. The two bile acids did not interfere in the transport of dexamethasone. However, SLS caused the Caco-2 cell junctions to become leakier, even at the sub-CMC 1 mM level. Also, the permeability of dexamethasone decreased at 10 mM SLS. 

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

Caco-2 assay permeabilities corrected for the UWL usually include Pu determined as a function of the stirring speed (since the cells are not stable over a wide pH range), as in Eq. (7.57) [511-514,552,578]... 

The trend in the industry has been to automate the Caco-2 permeability assay using semi- or fully automated procedures. With such a system it is possible to obtain a throughput in order of approximately 400-500 compounds per week. Automated Caco-2 assay systems are commercially available through Tecan/BD Bioscience and Bohdan Mettler Toledo. In addition, automated systems for maintenance of cell cultures are commercially available, while totally automated systems for both maintenance and culturing of cells grown on permeable filter supports are under development, e.g., by companies such as The Automation Partnership. 

Here, we briefly describe the automated Caco-2 assay used at the research site in AstraZeneca R D Molndal. The solubility of the test compounds is measured (or theoretically predicted) before they are run in the Caco-2 assay. In order to be able to make correct determinations of the permeability coefficient, the substance must be dissolved when added to cell monolayer in the transport experiment. Compounds with insufficient solubility are therefore not tested. We generally apply a test concentration of 10 pM, but in specific projects or under certain circumstances a concentration of only 1 pM is applied. The test compounds are first prepared in DM SO solution (1 mM) on a parent plate and are then diluted in transport buffer to give a final drug concentration of 10 pM (solution containing 1% DMSO) when added to the cell monolayers. 

The human colon adenocarcinoma cell line (Caco-2) assay is still commonly used to measure the potential for a compound to be absorbed. It measures the permeability potential because permeability is a component of the absorption process,3 27 31 Multiple reports discuss the use of HPLC/MS/MS to support the Caco-2 assay.32 38... 

In vitro assays are increasingly being used. Some of the reasons are cost, availability of more rapid results, and avoidance of negative publicity. Assays such as cytochrome P-450 enzymes, the Ames test, and the mouse lymphoma tk test are in vitro methods. For absorption studies, Caco-2 (Exhibit 5.9) and Madin-Darby canine kidney cell assays are now routinely used. Hepatocyte cell lines with metabolism capacity are being developed to test drug metabolism and toxicity. All these examples show that, where possible, pharmaceutical firms are gradually dispensing with animal studies. 

Preliminary pharmacokinetic behavior can be tested through a number of whole cell assays. Most commercially successful drugs are administered orally, meaning the drug must be able to enter the bloodstream by crossing membranes in the intestines. The most common membrane permeability assay is performed by monitoring the absorption and secretion of a compound by colon carcinoma cells (Caco-2). Diffusion across Caco-2 cell membranes is considered to be a valid model for molecular transport in the small intestines.16... 

Caco-2 cells are currently used at all levels of pharmaceutical research and development. Automation technologies allow tissue culture labs to easily maintain a large number of Caco-2 cells as well as to perform numerous permeability studies without the introduction of many common human errors. Combinatorial chemistry provides vast arrays of compounds, and Caco-2 assays can be used to assess potential permeability and metabolic issues before much money is invested in the candidate.6... 

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