MDCK permeability

Sawada and coworkers [25-27] studied the iso-pH 7.4 MDCK permeabilities of very lipophilic molecules, including chlorpromazine (CPZ) [25], These authors included 3% wt/vol bovine serum albumin (BSA) on the apical (donor) side, and 0.1-3% BSA on the basolateral (acceptor) side, and found that plasma protein binding greatly affected the ability of molecules to permeate cellular barriers. They observed cell tissue retention of CPZ ranging from 65 to 85%, depending on the... 

Taub, M. E., L. Kristensen, and S. Frokjaer. Optimized conditions for MDCK permeability and turbidi-metric solubility studies using compounds representative of BCS classes I-IV, Eur. J. Pharm. Sci. 2002, 15, 331-340... 

To meet the need of conducting HTS for ADME-Tox properties, many slow and expensive in vivo ADME assays are now being replaced by in vitro cell models. For intestinal absorption, Caco-2 cell lines and Madin Darby canine kidney (MDCK) cell lines are widely used to predict the absorption rate of candidate drug compounds across the intestinal epithelial cell barrier. A number of models for Caco-2 cell permeability and MDCK cell permeability have been reported that predict the oral absorption properties of drugs, mostly limited to small organic molecules. Caco-2 and MDCK permeability are related to "A" and "D" in the ADME-Tox. 

The known variability associated to the behavior of Caco-2 cell lines, together with the need to find a more robust and easy to grow type of culture led the pharmaceutical industry to substitute this system by, for example, MDCK cell cultures. So in silico modelers have also turned towards this new kind of in vitro cell data, although to a lesser extent than with Caco-2 systems. Perhaps the only notable example of the use of MDCK data is Refsgaard s categorical model, where a dataset of Caco-2 permeability values was enriched with MDCK permeability data [99]. 

Absorption / Gut Transport Caco-2 (MDCK) Permeability Gauges ability to cross gut wall... 

The MDCK permeability is the apparent permeability in the A—B direction in mulddrug resistant gene (MDR-l)-transfected MDCK ceUs in the presence of a inhibitor (10 pM cyclosporine A). 

The Madin-Darby canine kidney (MDCK) cell model is another commonly used cell monolayer system for the assessment of human intestinal absorption. MDCK cell lines offer a couple of key advantages over Caco-2 cells, including the ability to reach full differentiation in 3-7 days (as opposed to 21 days for Caco-2) as well as the ability to be co-transfected with Pgp, the primary intestinal efflux transporter. Good correlation has been observed between MDCK permeability and human absorption. " The primary disadvantage of the MDCK ceU line is that it is derived from canine, rather than human, cells and, as such, expression of transporters and metaboUzing enzymes is not identical. 

Irvine JD, Takahashi L, Lockhart K, Cheong J, Tolan JW, Selick HE, et al. MDCK (Madin-Darby canine kidney) cells a tool for membrane permeability screening. J Pharm Sci 1999 88 28-33. 

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

Recombinant CYP450s (also for safety assessment of drug-drug interactions) Permeability/absorption Caco-2 cells MDCK cells... 

Typical early in vitro permeability assessments measure the rate of flux of a compound from one side of a barrier to another [54, 55]. The barrier has historically been derived from a cell line, most commonly Caco-2 or Madin-Darby canine kidney (MDCK) cells. In the last several years, there has been substantial work and significant progress in the development of parallel artificial membrane permeability... 

Figure 8 shows PAMPA data for a subset of compounds from the two series in Fig. 7. The potential concern about low permeability for Series D is confirmed between the MDCK and PAMPA data. Series C, with the exception of one compound, also demonstrates good correlation between MDCK and PAMPA permeability the compound with low PAMPA permeability should be further analyzed for relevant structure-permeability information.

Fig. 7 MDCK A to B apparent permeability vs clogD. Filled squares and open triangles represent two different chemical series (series C and D, respectively)...

Since the widely accepted in vitro permeability model in the pharmaceutical industry is based on the use of cultured cells, such as Caco-2 or MDCK, it was appropriate to analyze the regression correlation coefficients based on the comparisons of Caco-2 log Pe and the log Pe values based on the human jejunal measurements [56]. 

Table 7 Permeability Coefficients of the Paracellular Route of Unperturbed and Cytochalasin D-Perturbed MDCK Cell Monolayers at 25°C... 

To estimate the relative importance of the tight junction and the lateral space composing the paracellular route, let us consider the permeability of mannitol across Caco-2 and MDCK cell monolayers. The results taken from earlier examples are presented below ... 

Interestingly, the permeability coefficients of mannitol in the two cell types are identical, most probably for different reasons, since the physical dimensions of the Caco-2 and MDCK monolayers (Table 8) are markedly different. Compared to the MDCK cell monolayer, the Caco-2 cell monolayer has a taller cell height, a shorter length in tight junctions, longer tortuous path lengths, and smaller slit width in lateral space. One recognizes that... 

Table 9 Theoretical Assessment of Lateral Space and Tight Junction Contributions to the Paracellular Permeability of Mannitol in Caco-2 and MDCK Cell Monolayers3...

Given the low permeability of the antioxidant across MDCK cell monolayers and its large membrane partition coefficient, efflux kinetic studies using drug-loaded cell monolayers cultured on plastic dishes could yield useful information when coupled with the following biophysical model. The steady-state flux of drug from the cell monolayer is equal to the appearance rate in the receiver solution ... 

Table 19 Comparison Between Permeability Coefficients of the Apical and Basolateral Membranes from Independent Efflux Kinetics from Drug-Loaded MDCK Cell Monolayers...

The initial conditions are CD = CD(0) at t = 0 and CR = 0 at t = 0. Efforts to obtain analytical solutions are tedious and unnecessary. By applying the change in concentrations (or mass) in the donor and receiver solutions with time to the Laplace transforms of Eqs. (140) and (141), the inverse of the simultaneous transformed equations can be numerically calculated with appropriate software for best estimates of a, (3, and y. It is implicit here that P Pap, Pbh and Ke are functions of protein binding. Upon application of the transmonolayer flux model to the PNU-78,517 data in Figure 32, the effective permeability coefficients from the disappearance and appearance kinetics points of view are in good quantitative agreement with the permeability coefficients determined from independent studies involving uptake kinetics by MDCK cell monolayers cultured on a flat dish... 

To reach such a site, a molecule must permeate through many road blocks formed by cell membranes. These are composed of phospholipid bilayers - oily barriers that greatly attenuate the passage of charged or highly polar molecules. Often, cultured cells, such as Caco-2 or Madin-Darby canine kidney (MDCK) cells [1-4], are used for this purpose, but the tests are costly. Other types of permeability measurements based on artificial membranes have been considered, the aim being to improve efficiency and lowering costs. One such approach, PAMPA, has been described by Kansy et al. [5],... 

Some laboratories have found an alternative to the short-term cultures by using cell lines other than Caco-2 cells. The most popular of these is Madin-Darby canine kidney (MDCK) cells, an epithelial cell line from the dog kidney. MDCK cells have been suggested to perform as well as Caco-2 cells in studies of passive drug permeability [56]. These cells have also been used to optimise the conditions for studies of low-solubility drugs [53]. However, as noted previously, the active transport processes of this cell line can be quite different to those of Caco-2 cells [28-30], Another cell line that only requires short-term culture is 2/4/A1, which is a conditionally immortalised rat intestinal epithelial cell line [86]. The 2/4/A1 cell line is discussed in Section 4.3.2.2 below. 

There are several approaches to estimating absorption using in vitro methods, notably Caco-2 and MDCK cell-based methods or using methods that assess passive permeability, for example the parallel artificial membrane permeation assay (PAMPA) method. These are reviewed elsewhere in this book. The assays are very useful, and usually have an important role in the screening cascades for drug discovery projects. However, as discussed below, the cell-based assays are not without their drawbacks, and it is often appropriate to use ex vivo and/or in vivo absorption assays. 

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